Mapping the Interactome of Saccharomyces cerevisiae ABC ......Two interactors were identified for...

Mapping the Interactome of Saccharomyces cerevisiae ABC

Transporters Pdr12p and Ste6p

by

Dunja Damjanovic

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Graduate Department of Molecular Genetics

University of Toronto

copy Copyright by Dunja Damjanovic 2010

ii

Mapping the Interactome of Saccharomyces cerevisiae ABC Transporters Pdr12p

and Ste6p

Dunja Damjanovic

Master of Science

Department of Molecular Genetics


2010

ABSTRACT

The ATP binding cassette (ABC) transporters represent the largest family of

transmembrane proteins and play important roles in human inherited disease such as the

multi-organ disease cystic fibrosis and cholesterol transport disorder Tangierrsquos disease

These proteins are also implicated in conferring multidrug resistance rendering many

cancer therapies ineffective as well as contributing to the pathogenicity of some

organisms The yeast ABC proteins Pdr12p a weak acid efflux pump and Ste6p the a-

factor exporter were screened for interacting partners using the integrated membrane

yeast two-hybrid (iMYTH) system to gain further insight into their biological function

Two interactors were identified for Ste6p however the Pdr12p screen identified 13 novel

interactions most notable of which are three other ABC transporters Pdr5p Pdr10p and

Pdr11p Subsequent functional analysis of double deletion mutants supports a genetic

interaction between Pdr12p and Pdr10p as the pdr12Δ pdr10Δ strain showed resistance to

increasing concentrations of weak organic acids

iii

ACKNOWLEDGMENTS

I wish to express my appreciation and gratitude to my supervisor Dr Igor Stagljar

for giving me the opportunity to work for him and learn from him I will always be

grateful for his advice which he gave freely for always listening to my concerns of

which there were many and most of all for pushing me beyond my limits and teaching

me never to give up

I would like to give my sincerest thanks to my committee members Drs Brenda

Andrews and Leah Cowen for their guidance throughout the years Their suggestions

and criticisms pushed me to continuously strive to improve and made me challenge

myself I am a wiser person for it

During my time here I have had the pleasure of meeting many great people and

have been fortunate enough to work alongside most of them on a daily basis A big thank

you goes out to all my lab mates both past and present for making our lab a fun and

interesting environment to work in For giving me guidance with new experiments

always listening and providing insights on overcoming a roadblock Dr Jamie Snider

has been a great teacher support and a person I relied heavily on for a second opinion

His willingness to answer my many questions provide me with great feedback and help

me out when I was unsure of how to proceed is much appreciated Though he challenged

every one of my results it was always with good intentions and has made my science

just that much better Dr Saranya Kittanakom whose smiling face always welcomed my

woes has been an invaluable help during my co-IP experiments Her knowledge and

advice gave me hope that one day it would all work Dawn Edmonds has been a fountain

of information over the years Her patience in teaching me to dissect tetrads and ordering

things for me on short notice is greatly appreciated I would also like to thank Dr Susan

Michaelis for her quick e-mail responses and advice on Ste6p

I would not be where I am today without the support both financial and

emotional of my parents and brother Mom and Dad thank you for always believing in

me for showing me that hard work pays off and for handling my being away from home

so well though I think Srdjan took it a little too well Your guidance throughout my life

has made me the person I am today and I will always appreciate that you always stood

behind everything I did and still wish to do

To my two best friends Dijana and Vanja I know that you often didnrsquot

understand what I did but I thank you for willing to try Most importantly I appreciate

you both listening to the ups and downs I encountered daily and for taking my mind off

such things and making me laugh whenever we were together or on the phone

Finally I want to give a big thanks to Tanja Durbic and Dr Katarina Vukojevic

for making my last few months fun and amusing for the random medical advice and the

many entertaining outings

Dunja Damjanovic

iv

Family that dear octopus from whose tentacles we

never quite escape nor in our inmost hearts ever quite wish to

ndash Dodie Smith

To my wonderful parents Miladin and

Gordana Damjanovic and my

brother Srdjan

v

TABLE OF CONTENTS

ABSTRACT ii

ACKNOWLEDGMENTS iii

LIST OF TABLES viii

LIST OF FIGURES ix

APPENDICES x

ABBREVIATIONS xi

INTRODUCTION 1

11 ABC Transporter Proteins 2

12 Yeast as a Model Organism 3

13 ABC Transporter Proteins in Saccharomyces cerevisiae 4

14 ABCG (PDR5) Subfamily 6

15 ABCB (MDR) Subfamily 8

16 The Other Yeast Subfamilies 9

17 Yeast Pdr12p 10

171 Protein and Function 10

172 Role in Food Spoilage 10

173 Known Interactions 12

18 Yeast Ste6p 13


182 Mating MAPK Pathway 13


19 Studying Protein-Protein Interactions (PPIs) 16

191 The Importance of PPIs 16

192 Yeast two-hybrid Technologies and their Limitations 16

193 Analysis of Membrane Protein Interactions 18

110 Ubiquitin and the MYTH Technology 19

1101 Ubiquitin and its Role in Protein Degradation 19

1102 Reconstitution of Split Ubiquitin 20

1103 The MYTH Technology 21

111 Thesis Rationale 24

MATERIALS AND METHODS 25

21 Yeast Strains Media and Growth Conditions 26

22 Generation of Endogenously CT- and CYT-tagged Bait Proteins 26

vi

23 Construction of the Prey Random Genomic DNA and cDNA Libraries 26

24 Verifying Proper Localization of CYT-tagged Bait Proteins 26

25 NubGNubI Test 27

26 Verification of C(Y)T-tagged Bait Functionality 28

261 Generation of Deletion Mutants 28

262 Verifying Deletion Mutants 28

263 Verifying Pdr12-C(Y)T Function 29

264 Verifying Ste6-C(Y)T Function 29

27 The iMYTH Assay 30

271 Large Scale Transformation 30

272 Patching and Recovering Putative Interactors 31

273 Amplification and Recovery of Prey Plasmid DNA 31

274 Prey Identification 32

275 Bait Dependency Test 32

28 Generation of Double Deletion Mutants 33

29 Generating Full-length tagged Pdr5p Pdr10p and Pdr11p 34

291 Gap Repair Method 34

292 Gateway Cloning 35

210 Functional Assays for Pdr12p 36

2101 Spot Assays 36

2102 Liquid Panelling Assay 37

2103 Co-Immunoprecipitating Interacting Proteins of Pdr12p 37

2104 Western Blot Analysis 38

211 Extending Ste6p Duration at the Plasma Membrane 39

RESULTS 40

31 Endogenously CT and CYT-tagged Bait Proteins Successfully Generated 41

32 CYT-tagged Integrated Bait Proteins Strains Localize Correctly 41

33 Tagged Bait Strains Pass NubGNubI Test 42

34 Functional Analysis of Bait Proteins 43

341 Pdr12-CT Grows in the Presence of Sorbic Acid 43

342 Ste6-CT is Able to Mate 44

35 iMYTH Screening Results 45

351 Large Scale Library Transformation 45


vii

353 Pdr12p Interactome 47

354 Ste6p Interactome 50

36 Generation of Double Deletion mutants with pdr12Δnat 50

37 pdr10Δkan pdr12Δnat Mutant Shows Resistance to Weak Acids 53

371 Spot Assays 53

372 TECAN Liquid Growth Assay 54

38 A Variety of Drugs Have no Affect on the Double Deletion Mutants 58

381 Spot Assays 58


39 Increasing Ste6p Duration at the Plasma Membrane 61

391 Treatment with α-factor 61

3102 Deletion of SAC6 63

DISCUSSION 65

41 GO Analysis 66

42 Protein Interactions of Interest 66

421 iMYTH Identifies an Interaction Between Pdr12p and Pdr5p 66


423 iMYTH Identifies Pdr11p as a Novel Interactor of Pdr12p 69

424 Vps9p is a Novel Interactor of Ste6p 70

43 Poor Detection of Ste6p Interactions 71

44 Putative Role for Pdr10p in the Weak Acid Response 72

45 Lack of Expression of Prey Proteins 74

46 iMYTH as a System for the Detection of PPIs 75

FUTURE DIRECTIONS AND CONCLUSIONS 77

51 Concluding Remarks and Future Directions 78

REFERENCES 84

APPENDIX 91

viii

LIST OF TABLES

Table 1 iMYTH Screening Results for Pdr12p and Ste6p

Table 2 Summary of Double Deletion Strains

Table 3 Yeast strains used in this study

Table 4 Plasmids used in this study

Table 5 Primers used in this study

Table 6 PCR Reactions

Table 7 PCR Programs

Table 8 iMYTH Identified Prey Protein Regions of Interaction from Pdr12p Screen

Table 9 iMYTH Identified Prey Protein Regions of Interaction from Ste6p Screen

Table 10 Description of Pdr12p Interactors

Table 11 Description of Ste6p Interactors

ix

LIST OF FIGURES

Figure 1 ABC transporter structure

Figure 2 Phylogenetic tree of yeast ABC proteins

Figure 3 Subcellular localization of Saccharomyces cerevisiae ABC transporters

Figure 4 Saccharomyces cerevisiae mating MAPK signalling pathway

Figure 5 Schematic of the iMYTH system

Figure 6 CYT-tagged bait protein localization

Figure 7 NubGNubI test for integrated bait strains

Figure 8 CT tag does not interfere with Pdr12p function

Figure 9 Evaluating Ste6-CT function with a mating assay

Figure 10 An example of a bait dependency test

Figure 11 Pdr12p Interactome

Figure 12 Ste6p Interactome

Figure 13 Weak acid stress assay

Figure 14 Sorbic acid liquid growth assay

Figure 15 Benzoic acid liquid growth assay

Figure 16 Drug sensitivity assay

Figure 17 Haloperidol liquid growth assay

Figure 18 Ste6-CYT treatment with α-factor

Figure 19 Ste6-CYT sac6Δnat localization

Figure 20 Pdr12p Bait Dependency Test

Figure 21 Ste6p Bait Dependency Test

Figure 22 Sorbic and benzoic acid liquid growth assay replicate

x

APPENDICES

Appendix I Yeast Strains Media Recipes and Reagents

Appendix II PCR Protocols and Primer Sequences

Appendix III Sequences of Pdr12p Identified Interactors

Appendix IV Pdr12-CT Bait Dependency Test

Appendix V Sequences of Ste6p Identified Interactors

Appendix VI Ste6-CT Bait Dependency Test

Appendix VII Definitions of Pdr12 and Ste6p iMYTH Identified Interactors

Appendix VIII Weak Acid Liquid Growth Assay Replicate

xi

ABBREVIATIONS

ABC ndash ATPndashbinding cassette

AD ndash Activation domain

ATP ndash Adenosinetriphosphate

Cub ndash C-terminal half of ubiquitin

CYT tag ndash Cub-YFP-TF tag

DBD ndash DNA binding domain

DUBsUBPs ndash Deubiquitinating enzyme(s)Ubiquitin-specific protease(s)

ER ndash Endoplasmic reticulum

FeS ndash Iron-sulfur

iMYTH ndash Integrated membrane yeast two-hybrid

Kan ndash Kanamycin

MSDTMD ndash Membrane spanning domainTransmembrane domain

MAPK mitogen activated protein kinase

Nat ndash Nourseothricin acetyl transferase

NBD ndash Nucleotide binding domain

Nub ndash N-terminal half of ubiquitin

NubI ndash Wildtype N-terminal half of ubiquitin

NubG ndash Mutant N-terminal half of ubiquitin

ORFs ndash Open reading frame(s)

PCR ndash Polymerase chain reaction

PDR ndash Pleiotropic drug resistance

PM ndash Plasma membrane

PPIs ndash Protein-protein interaction(s)

PURE ndash Phosphorylation ubiquitination recognition and endocytosis

RRS ndash Ras recruitment system

TF ndash Transcription factor

tMYTH ndash Traditional membrane yeast two-hybrid

WARE ndash Weak acid response element

WT ndash Wildtype

Y2H ndash Yeast two-hybrid

YFP ndash Yellow fluorescent protein

CHAPTER 1

INTRODUCTION

2

11 ABC Transporter Proteins

Survival at the cellular level is dependent on the ability of the cell to regulate the

selective passage of molecules and ions across its membranes not only for the acquisition

of nutrients and the excretion of waste products but for various regulatory and signalling

functions as well (1 2) Movement across the cellular membranes for the mentioned

processes is mediated by specialized proteins called transporters ATP-binding cassette

(ABC) transporters represent a large evolutionarily conserved family of integral

membrane proteins (1) currently estimated to consist of more than 3000 members (3)

These proteins are central to many physiological processes (4) and use the binding and

hydrolysis of ATP to power the translocation of a diverse assortment of substrates against

their concentration gradients across cellular membranes (1)

ABC transporters are ubiquitous in all organisms from bacteria to man and exist

as both exporters which can be found in both prokaryotes and eukaryotes and importers

which are exclusive to prokaryotic organisms (1) These proteins share a conserved

architecture known as the ABC core consisting of two homologous halves each

containing a membrane spanning domain (MSD) which is involved in substrate

specificity and a nucleotide-binding domain (NBD) which together form a ldquofull-lengthrdquo

functional transporter (1 2 4 5) (Fig 1) The NBD binds ATP and couples its

hydrolysis to substrate transport which is critical for ABC protein function (5) This

domain also has several conserved regions including the Walker A and B motifs and the

ABC signature motif LSGGQ (1 5)

3

Figure 1 ABC transporter structure Shown here is a standard arrangement for a full-length transporter

protein which consists of two hydrophobic MSDs and two NBDs The MSDs typically but not always

span the membrane six times while the NBD are responsible for ATP binding and hydrolysis and are

located in the cytoplasm

ABC transporters play an important role in many human diseases and

physiological processes (4) such as maintaining the blood-brain barrier which prevents

access of cytotoxic drugs to the brain and mediating cellular resistance to

chemotherapeutic drugs (5) Loss-of-function mutations in the genes encoding ABC

transporter proteins are implicated in a variety of human inherited diseases such as cystic

fibrosis Tangierrsquos disease and Stargardtrsquos muscular dystrophy among others (4 5) The

overexpression of ABC proteins leads to multidrug resistance in pathogenic

microorganisms as well as mammalian cells as is seen in the human MDR1 protein

which is able to expel almost all known anticancer drugs conferring resistance to tumor

cells (4 5) as a result hindering treatment and cancer therapy

Given their prevalence in all life forms ABC transporter proteins are of particular

interest to the scientific community both for their implications in human health and their

potential as therapeutic targets in treating cancer and preventing multidrug resistance

12 Yeast as a Model Organism

Over the years Saccharomyces cerevisiae being a simple eukaryote that can easily be

manipulated has emerged as an important tool for the study of eukaryotic cell function

The biochemical biological and genetic tractability of yeast make it an ideal model

4

system for studying protein interaction networks and function as well as for defining

cellular pathways (5) Yeast is also a very practical organism to work with as it is

inexpensive to maintain grows quickly and is safe when handled properly The genome

of yeast is fully sequenced which has facilitated the construction of the yeast deletion

collection providing yet another resource for the analysis of phenotypes and genetic

interactions under a variety of conditions In addition to a versatile and straightforward

transformation system (6) a number of powerful genetic and molecular approaches that

use yeast have been developed some of which can readily be automated facilitating

high-throughput studies (7) Finally many genes implicated in human diseases and

multidrug resistance have homologues in yeast It is also important to note that yeast and

human genomes share high homology which allows conclusions from the study of yeast

to provide insight into the physiological and biochemical mechanisms of human

homologues (8)

13 ABC Transporter Proteins in Saccharomyces cerevisiae

With the completion of the yeast genome sequence project in 1996 Saccharomyces

cerevisiae became the first organism for which the complete inventory of ABC

transporter proteins was available (5) It is estimated that close to 30 of the yeast

proteome consists of membrane proteins 10 of which are believed to be responsible for

the transport of small molecules through the plasma membrane (PM) (9) The yeast

genome encodes 30 ABC transporter proteins originally identified from BLAST searches

for homologues of the NBD1 of STE6 Of these proteins 22 are predicted to be true

ABC transporters while the remaining eight are believed to have regulatory roles as

opposed to transport functions due to the fact that they do not have any predicted

membrane spans (5 10) Based on phylogenetic analysis the 22 yeast ABC transporters

5

have been divided into six subfamilies (Fig 2) which have recently been renamed

following the mammalian nomenclature replacing the yeast subfamily names of MDR

MRPCFTR ALDP RLI YEF3 and PDR5 with ABCB to ABCG respectively (5)

Figure 2 Phylogenetic tree of yeast ABC proteins Members of the same subfamily are indicated by

grouping under the same coloured arc Subfamily names are indicated outside of the arc in the

corresponding colour following mammalian nomenclature For each subfamily a mammalian member

was used in the analysis as a point of reference These are indicated by an ldquohrdquo before their name The

asterisk indicates yeast proteins that are not closely homologous to any of the mammalian transporter

subfamilies The ABCA subfamily is absent in yeast Based on Paumi et al (5)

The majority of yeast ABC proteins localize to the plasma membrane where they

are responsible for the efflux of many substrates however these proteins are also found

within the membranes of intracellular organelles (5) As can be seen in Fig 3 the

peroxisome mitochondria and vacuole of a yeast cell all have several ABC proteins

6

within their membranes however no ABC proteins localize to the nucleus or

endoplasmic reticulum (ER) (5)

Fungal ABC proteins are involved in a variety of cellular functions from clinical

drug resistance development and translation elongation to cellular detoxification and

stress response (11) In addition to having a wide substrate specificity with respect to

drug transport ABC proteins also mediate the translocation of ions heavy metals amino

acids carbohydrates and even whole proteins across cellular membranes (11)

Figure 3 Subcellular localization of Saccharomyces cerevisiae ABC transporters The 22 yeast ABC

proteins are found in the membranes of organelles of the cell and the PM With the exception of Ste6p

(ABCB) and Yor1p (ABCC) all of the ABC proteins found within the PM belong to the ABCG subfamily

Pxa1p and Pxa2p belong to the ABCD subfamily the mitochondrial transporters are ABCB members

while the vacuolar transporters make up the rest of the ABCC subfamily P designates peroxisome V the

vacuole M the mitochondria N the nucleus and ER the endoplasmic reticulum Transporters belonging to

the same subfamily are indicated by colour Two cylinders indicates a full-length transporter while one

cylinder indicates a half-sized transporter Based on Jungwirth and Kuchler (3) and Paumi et al (5)

14 ABCG (PDR5) Subfamily

In addition to being divided into subfamilies eukaryotic ABC proteins have also been

subdivided into either full or half length transporters (12) The mammalian ABCG or

White subfamily consists of five unique half transporters named ABCG1 ABCG2

7

ABCG4 ABCG5 and ABCG8 These proteins have a peculiar domain organization with

the NBD at the N-terminus followed by the MSD (12-14) In order to become fully

functional transporters they form homodimers (ABCG1 ABCG2 and ABCG4) or

obligate heterodimers (ABCG5 and ABCG8) (12 14) With the exception of ABCG2 all

members of this family play a significant role in the transport of sterols (12) especially

the efflux of cholesterol (14) The altered expression andor activity of both ABCG2 and

the heterodimer ABCG5ABCG8 has clinical relevance Altered ABCG2 results in

resistance to chemotherapy while changes in the heterodimer result in sitosterolemia

which is characterized by an accumulation phyto- and shellfish sterols (12 14)

Previously known as the PDR5 subfamily the Saccharomyces cerevisiae ABCG

subfamily with its 10 members is the largest and best characterized of all the yeast ABC

subfamilies to which Pdr12p belongs With the exception of Adp1p all protein members

are classified as full length transporters and are involved in a variety of functions

including metal ion resistance (15) and efflux of weak organic acids (16) All members

of this subfamily reside in the PM (Fig 3) Perhaps some of the most extensively studied

and best characterized members of this family include Pdr5p and Snq2p (17 18) Both

proteins mediate multidrug resistance through ATP-dependent efflux (15) and are able to

recognize numerous structurally and functionally unrelated compounds (18) In addition

to sharing high homology with one another (15) these proteins have largely overlapping

substrate specificity (18 19)

Pleiotropic drug resistance (PDR) in yeast is homologous to multidrug resistance

(MDR) observed in parasites bacteria fungal pathogens and mammalian tumor cells (3

11 20) Resistance to multiple cytotoxic compounds is an acquired trait (21) with the

8

major determinants mediating this resistance being ABC transporter proteins (17) PDR

results from the overexpression of membrane proteins that mediate drug efflux from the

cell which can occur through mutations in genes encoding the proteins or their

transcriptional regulators (3 22) With a large number of these proteins in the PM which

constitute the first line of defence against harmful compounds (23) yeast can quickly

counteract substrate toxicity through the PDR network of proteins (3) This acquired

resistance poses major challenges for cancer therapy and the treatment of infectious

diseases as well as the development of effective therapeutics (22 23)

Several proteins in this family are responsible for mediating acquired multidrug

resistance (15 18) while on the other end of the spectrum Pdr12p another member of

this family that acts as a weak acid anion pump has important implications for the food

industry specifically the preservation of food products and beverages (19 24)

15 ABCB (MDR) Subfamily

This subfamily of yeast proteins only comprises of four members three of which reside

in the inner mitochondrial membrane and are considered half length transporters (5)

while Ste6p is localized to the PM (Fig 3) (19) and is a full length transporter protein (5)

Ste6p is required for mating of yeast cells as it is responsible for the transport of the

mating pheromone a-factor out of the cell (11) Atm1p acts as a homodimer (25) and

exports iron-sulfur (FeS) clusters from the mitochondria and as such plays an essential

role in the generation of cytosolic FeS proteins (26) Mdl1p is responsible for the export

of mitochondrial peptides generated by proteolysis (27) is a suppressor of Atm1p and

also has a role in the regulation of cellular resistance to oxidative stress (28) While

Mdl2p is highly similar to Mdl1p at the sequence level it does not play a role in the

export of peptides and its function remains unknown (29)

9

16 The Other Yeast Subfamilies

The second largest yeast subfamily of ABC transporters with six members is the ABCC

subfamily All six of these proteins have the typical structural organization and share

significant homology with the human multidrug resistance-associated protein 1 (MRP1)

and the cystic fibrosis chloride channel protein (CFTR) (11) both of which have clinical

importance These proteins function as vacuolar detoxification pumps and mediate both

multidrug and heavy metal resistance (11 30) With the exception of Yor1p which

localizes to the PM (3) all other proteins of this subfamily are found in the vacuolar

membrane (Fig 3) (3 11 31) One of the most extensively studied members of this

subfamily is Ycf1p the yeast cadmium factor which mediates vacuolar detoxification of

heavy metals and xenobiotics by transporting them as glutathione-S conjugates (11 32)

Ycf1p is also responsible for the accumulation of red pigment in ade2 mutant cells (3

32) The other well characterized protein from this subfamily is Yor1p whose deletion

mutants though viable are hypersensitive to oligomycin and reveromycin A (11) as well

as other xenobiotics (11 33)

The ABCD subfamily is comprised of two half-sized transporters Pax1p and

Pax2p located in the peroxisomal membrane (Fig3) (3 11) Both proteins have one

MSD that spans the membrane six times and a single NBD In addition Pax1pPax2p

are orthologues of the human Pmp70 and ALDp-like peroxisomal transporters associated

with the fatal neurodegenerative disease adrenoleukodystrophy (3 11)

The ABCE and ABCF subfamilies in yeast have one and six members

respectively all of which lack MSDs and have not been studied with the exception of

two members of the ABCF subfamily Yef3p and Gcn20p (11) Yef3p is believed to

function as an elongation factor and is encoded by the only essential ABC gene In

10

addition its overexpression causes hypersensitivity to the translational inhibitors

paromomycin and hygromycin B Though as of yet unconfirmed a possible role for

Gcn20p could be the regulation of amino acid utilization (11)

There are also two proteins Caf16p and Ydr061Cp that have not yet been

classified as their sequences are more distantly related to the other ABC transporter

proteins (11) and are not close homologues of any mammalian subfamily member (5)

Though they do have a NBD with degenerate ABC signature motifs these proteins still

lack predicted membrane spanning regions (11)

17 Yeast Pdr12p

171 Protein and Function

The yeast PDR12 gene encodes a 1511 amino acid long 171 kDa ABC transporter

protein that resides in the PM (Fig3) (3) The protein is a full length transporter with

(NBD-MSD6)2 topology arranged in the reverse conformation The promoter region of

Pdr12p contains a cis-acting weak acid response element (WARE) required for the

binding of the transcription factor War1p (34) In the presence of weak organic acids

such as sorbic and benzoic acid Pdr12p becomes strongly induced causing an increase

of the protein to accumulate at the PM (24) The induction of PDR12 is rapid mainly

regulated at the level of transcription and is specific for weak acid stress (34) This

protein is the first ABC transporter to be assigned the function of a weak acid anion pump

(16) and is essential for the adaptation and growth of cells in the presence of weak acid

stress (35) as is the phosphorylation activation and DNA binding of War1p (36)

172 Role in Food Spoilage

Weak acids have a long history as additives in food and have primarily been used to

prolong the shelf life and preserve food quality through the inhibition of spoilage micro-

11

organisms (36 37) The most commonly used compounds in the food industry include

sulphites used in wine making (36) as well as the naturally occurring short-chain (C1-

C7) weak organic acids such as sorbate benzoate acetic and propionic acids used in

various foods and beverages (34) With respect to yeast weak acid preservatives

characteristically cause an extended lag phase and cell stasis as opposed to cell death

(24 36)

In solution weak acid preservatives exist in a pH-dependent equilibrium between

the undissociated and the dissociated states (35) They have optimal inhibitory activity at

lower pH values as this favours the undissociated uncharged state of the molecule

which is freely permeable across the PM (35) Once the acid molecule enters the cell it

encounters the higher cytoplasmic pH and dissociates into anions and protons which

being charged particles cannot cross the PM resulting in their accumulation within the

cell (34-36) The mechanism of growth inhibition by weak acid preservatives is not yet

fully understood however it is proposed that the accumulation of protons leads to

cytoplasmic acidification which in turn inhibits a number of important metabolic

processes including active transport glycolysis and signal transduction (36)

The ability of microbes to survive and grow in foods that contain preservatives is

largely due to their ability to adapt to stress (16) Yeasts that are major spoilage

organisms include Zygosaccharomyces as well as some isolates of Saccharomyces

cerevisiae (16) whose ability to grow in the presence of the maximum permitted levels

of preservatives causes severe economic losses and poses potential health hazards (37)

The ability of Saccharomyces cerevisiae to grow in the presence of sorbic and benzoic

acids involves the induction on the efflux pump Pdr12p whose active efflux of acid

12

anions from the cell results in adaptation of weak acid induced stress (16 20) Through

this function Pdr12p is able to neutralize the intracellular environment rendering any

inhibitory activity of the weak acid preservative futile allowing normal metabolic

processes to continue unhindered

As Pdr12p is implicated in the spoilage of food insight into the function of this

protein and how it renders yeast resistant to preservatives has important implications for

the food industry By identifying interacting partners the exact mechanism mediating

this weak acid resistance could be elucidated and with a greater understanding of this

process new methods with the ability to obstruct the cells resistance to food preservatives

can be developed avoiding economic losses and potential health risks associated with

spoiled food products

173 Known Interactions

According to the Saccharomyces Genome Database (SGD) Pdr12p has a total of 48

known physical interactions the majority of which were identified by a genome-wide in

vivo screen using the protein-fragment complementation assay (PCA) (38) Some of the

more notable interactions include Gpa2p the α-subunit of a G-protein and Hsp30p a

stress induced protein of the plasma membrane that negatively regulates the H(+)-

ATPase Pma1p In addition Pdr12p was shown to interact with proteins of the major

facilitator superfamily such as the sugar transporters Hxt1p and Hxt5p as well as the

multi-drug transporters Qdr2p and Qdr3p Most interestingly the PCA screen also

identified Snq2p and Yor1p as interactors of Pdr12p both of which are major drug

pumps belonging to the ABC superfamily the latter of which is also similar to the human

CFTR (38)

13

18 Yeast Ste6p


The first ABC transporter gene discovered in Saccharomyces cerevisiae was STE6 which

was subsequently shown to encode Ste6p a 1209 residue full length transporter protein

localized to the PM with forward (MSD6-NBD)2 topology (3 19) Perhaps one of the

best characterized yeast ABC transporters Ste6p is the exporter of the mating pheromone

a-factor (11) and is a close homologue of the human P-glycoprotein with which it shares

approximately 60 homology (39 40)

Despite its site of function being the PM Ste6p resides only briefly at the cell

surface with a half life estimated to be 15-20 minutes (41 42) Due to rapid and

constitutive endocytosis after which Ste6p is ultimately delivered to the vacuole for

degradation (11 43) the protein does not accumulate at the PM (42) It was shown that

Ste6p follows a complex trafficking pattern for the internalization of PM proteins that

involves phosphorylation ubiquitination recognition and endocytosis appropriately

named the PURE pathway (41) Likewise it was shown that ubiquitination is a critical

signal for the internalization of Ste6p (41 42) and as would be expected any mutations

that affect the ubiquitination process or any other step in the pathway result in the

stabilization of Ste6p at the plasma membrane (41 43)

182 Mating MAPK Pathway

Saccharomyces cerevisiae cells produce and respond to peptide hormones whose role is

to induce physiological processes that lead to the conjugation of two haploid cells

resulting in the formation of a diploid cell (44) Biologically active α-factor is produced

by MATα cells from specific proteolytic processing events that occur during transit of its

precursor molecule through the yeast secretory pathway which is its mode of release

14

from the cell (44) Unlike α-factor mature a-factor is a post-translationally modified

peptide processed and released from MATa cells (44) via the ATPase activity of Ste6p

(39) The STE6 gene product is essential for mating between yeast cells to occur and not

surprisingly its deletion results in a sterile phenotype (44 45)

Figure 4 Saccharomyces cerevisiae mating MAPK signalling pathway Proteins are shown as labelled

shapes black arrows indicate translocation or protein activation while T-bars indicate inhibition Protein

association is indicated by the double-headed black arrow The binding of a-factor pheromone by receptor

Ste2p causes dissociation of the heterotrimeric G-protein (1) into G subunit and the G dimer Upon

the dissociation of the G protein Ste4p recruits the MAPK scaffold Ste5p to the membrane (2) Ste5p

recruitment activates the MAPK cascade in which Ste20p Ste11p Ste7p and the MAP kinase Fus3p

phosphorylate one another in sequential order Phosphorylated Fus3p (3) translocates to the nucleus and

phosphorylates Dig1p and Ste12p eliminating Dig1p repression of Ste12p (4) Ste12p is then free to

activate transcription of pheromone-responsive genes Based on Elion (46)

The receptor-G-protein-coupled mitogen-activated protein kinase (MAPK)

pathway mediates the response of a cell to the presence of a pheromone (Fig 4) (46)

15

The binding of a-factor to its receptor Ste2p on the surface of a MATα cell induces

several cellular responses including the arrest of the cell cycle in G1 phase The binding

also causes the heterotrimeric G-protein to dissociate into a Gα subunit Gpa1 and the

Gβγ dimer Ste4-Ste18 Ste4p then helps to recruit the MAPK scaffolding protein Ste5p

to the membrane which activates the MAPK cascade a series of sequentially activated

protein kinases This ultimately leads to the transcriptional activation of pheromone-

responsive genes that allow individual cells to synchronize their cell cycles elongate and

form a projection toward their mating partner and finally fuse with one another to yield a

diploid cell (46 47)


Although Ste6p is involved in mating there are only 13 listed interactions on the SGD 7

of which are genetic interactions involving proteins of the 20S and 26S proteosome (48)

The remaining 6 physical interactions do not include proteins involved in mating and

have been detected using different methods Two of the proteins Ste6p interacts with are

Lsm4p and Lsm5p (49) which are believed to form heteroheptameric complexes and

thought to be involved in mRNA decay andor tRNA and rRNA processing Other

interactions include Sec72p (50) and the ER-associated protein Ssm4p (51) Perhaps one

of the more intriguing interactions is the one Ste6p has with itself It was shown that

STE6 half-molecules interact physically assembling in vivo to form a functional

transporter protein (52) The same was also demonstrated for a STE6 half-molecule and

full-length STE6 (52) however two full length Ste6p proteins were not shown to interact

Though the function of Ste6p is known the mechanisms behind it are not well

understood Given that only 6 proteins have been identified that physical interact with

Ste6p by identifying novel interacting partners of Ste6p further insight can be gained

16

into the mechanisms of transport and its internalization which could be applied to better

understand its homologue the human P-glycoprotein In addition novel roles for this

protein could be identified

19 Studying Protein-Protein Interactions (PPIs)

191 The Importance of PPIs

Protein-protein interactions (PPIs) are an essential aspect in every biological process as

they regulate many cellular functions including cell signalling metabolism regulation

and the formation of macromolecular structures (38 53 54) These interactions can also

confer specificity to the interactions occurring between an enzyme and its substrate and

are often involved in the channelling of substrates through the formation of multi-protein

complexes (54) Membrane proteins also play important roles in biological processes as

they control membrane permeability to countless structurally and functionally unrelated

compounds and are also involved in sensing chemical and physical stimuli from the

external environment such as hormones and pathogens (54) In addition membrane

proteins are of substantial therapeutic and diagnostic importance as it is estimated that

50 of currently known drug targets are membrane ion channel or receptor proteins (7

53) Insight into the function of a specific protein can be gained by examining the

proteins it can bind to and with the sequencing of entire genomes of representative

model organisms many genetic and biochemical methods have evolved to address the

technological challenges faced when investigating PPIs with the yeast two-hybrid (Y2H)

being the most popular

192 Yeast two-hybrid Technologies and their Limitations

First published in 1989 as an approach to detecting PPIs (55) the Y2H assay is one of the

most successfully and widely used methods for investigating PPIs in vivo (56 57) The

17

basic idea behind all two-hybrid methods is to split a protein into two halves that do not

function independently of one another but do so when brought together again In the

Y2H assay a protein of interest called the bait is fused to the DNA binding domain

(DBD) of a transcription factor (TF) while another protein called the prey is fused to

the activation domain (AD) of the same transcription factor (53 57 58) Both fusion

proteins are co-expressed in yeast where their interaction leads to the reconstitution of a

functional TF which activates reporter genes typically HIS3 LEU2 and lacZ allowing

for detection by growth on selective medium and a colour signal respectively (53 57

58)

Two-hybrid technologies are best suited for measuring direct interactions between

pairs of proteins (38) and since the Y2H is a genetic assay it is a system well suited for

high-throughput applications (58) Two of the best known adaptations of the Y2H

system for large-scale use are the matrix or array approach and the library screening

approach both of which have been successfully used for the generation of genome-wide

protein interaction maps in yeast (58) In the matrix approach yeast open reading frames

(ORFs) are amplified using the polymerase chain reaction (PCR) are cloned as both

fusions of the DBD and the AD and introduced into reporter strains of opposing mating

type A reporter strain expressing a DBD fusion is mated to all the different AD fusions

comprising the array and positive interactions are identified by the ability of diploid cell

to grow on selective medium The library screening approach uses complex libraries of

AD fusions containing both full length and fragmented ORFs which are divided into

pools used to mate with a strain expressing a DBD fusion bait protein Similarly diploid

strains containing an interacting pair are selected by their ability to grow on selective

18

medium (58) Both techniques have been used to study all 6000 ORFs to generate a

glimpse into the yeast interactome (59 60) and the Y2H technique has even been

adapted for the use in mammalian systems (61)

Though an effective rapid and easy to use system one that has been successfully

employed in the detection of more than 50 of interactions described in literature (58)

the Y2H assay is not without limitations Many naturally occurring PPIs cannot be

detected with this method due to the requirement of the system for the interacting

proteins to be located in the nucleus in order to activate the reporter genes (7) Therefore

any interaction between proteins outside of the nucleus cannot be detected Membrane

proteins in particular present a significant challenge for the Y2H methodology

Transmembrane proteins are anchored in the membrane and therefore form aggregates

outside of the membrane due to their highly hydrophobic and insoluble nature Using

soluble domains is an option but can affect the detection of certain interactions and as

such is not an ideal solution In addition membrane proteins can have post-translational

modifications or oligomerize through interactions involving their MSD neither of which

are favourable for the nuclear-based Y2H assay (7 57) Another serious challenge for

the Y2H assay is the frequent and high occurrence of false negatives and positives the

latter of which can range anywhere from 25-45 for a large-scale screen (53)

193 Analysis of Membrane Protein Interactions

To overcome the limitations of the Y2H system several genetic screening methods have

been developed to address the problem of investigating interactions involving membrane

proteins while retaining the advantages of the original Y2H assay These include the Ras

recruitment system (RRS) and the reverse RRS both of which are based on the Ras

pathway in yeast the G-protein fusion technology where the inactivation of the G-

19

protein signalling pathway serves as the readout (7 58) and the rUra3 based split-

ubiquitin system (58) Genetic assays that are based on the complementation of proteins

or protein fragments and allow for the monitoring of membrane protein interactions in

real time in organisms other than yeast have also been developed (7) These include the

β-galactosidase complementation assay dihydrofolate reductase (DHFR) assay and the β-

lactamase assay (7) Though all of these technologies are suitable for the study of

transmembrane proteins they still have limitations In the case of the RRS and reverse

RRS systems membrane proteins cannot be used as bait or prey respectively (7 58)

limiting the identification of interactions to only those that occur between membrane and

cytosolic proteins Though successfully used to demonstrate an interaction between two

defined interaction partners syntaxin 1 and Sec1 the G-protein based system has yet to

be used in large-scale library screening (7 58)

110 Ubiquitin and the MYTH Technology

Based on the ability of ubiquitin to reconstitute when split into two moieties the

membrane yeast two-hybrid (MYTH) system (62) was developed to overcome the

limitations of the traditional Y2H assay (55) specifically the inability of the assay to

investigate interactions involving membrane proteins and as such is a powerful tool for

the study of ABC transporter interacting partners

1101 Ubiquitin and its Role in Protein Degradation

Ubiquitin is a small highly evolutionarily conserved polypeptide comprised of 76

amino acid residues that is found in every living organism and serves as a signal for the

degradation of proteins (63) Degradation of a protein via the ubiquitin-mediated

proteosome pathway occurs in two steps the first of which tags the target substrate with

multiple ubiquitin molecules by covalent bond formation which is followed by the

20

degradation of the tagged protein by the 26S proteosome a large multicatalytic protease

Conjugation of ubiquitin to the substrate is a three step process that starts with the

activation of ubiquitin in an ATP driven reaction by the ubiquitin-activating enzyme E1

which generates a first thiol ester intermediate The ubiquitin-conjugating enzyme E2

transfers the activated ubiquitin moiety via an additional thiol ester intermediate from

E1 to E3 a member of the ubiquitin-protein ligase family The E3 catalyzes the covalent

attachment of ubiquitin to the substrate by forming an isopeptide bond between the

molecule and an internal Lys residue of the substrate A polyubiquitin chain is

synthesized by successively adding activated ubiquitin molecules to the internal Lys

residue of the previously conjugated ubiquitin and is recognized the 26S proteosome

complex On the other hand cell surface proteins such as G-protein coupled receptors

pheromone receptors and membrane proteins are mono ubiquitinated which results in

their internalization rather than degradation These proteins are ultimately shuttled to the

vacuole for degradation (63)

Degradation of cellular proteins is a highly complex and tightly regulated process

that plays important roles in a variety of pathways during cell life and death as well as

health and disease (63) The selective degradation of many eukaryotic proteins is carried

out by ubiquitin-mediated proteolysis (64) which as a system is key for maintaining

cellular quality control defence mechanisms and homeostasis (63 65) To name a few

ubiquitin-mediated proteolysis is involved in the process of cell cycle regulation and

division DNA repair and response to stress (63 64)

1102 Reconstitution of Split Ubiquitin

In 1994 it was discovered that when ubiquitin was split into a C-terminal moiety

termed Cub and an N-terminal moiety called Nub the two would spontaneously

21

reconstitute if expressed within the same cell to form a ubiquitin molecule that is

recognized by ubiquitin-specific proteases (UBPs) (66) In the same study it was also

shown that reconstitution of the two halves of ubiquitin would also occur when they were

expressed as fusions of proteins and that by mutating a single residue of Nub the

reconstitution of the molecule was abolished However if the proteins fused to the Cub

and Nub moieties interact in vivo ubiquitin can once again become reconstituted and its

subsequent cleavage by UBPs can be restored (66)

This discovery made it possible to study PPIs within a living cell and as a

function of time It also allows for the detection and analysis of larger protein

complexes weak and transient interaction and the study of interactions occurring

between membrane proteins and as such is an integral part of the MYTH system

1103 The MYTH Technology

In the traditional MYTH (tMYTH) system a membrane protein of interest the

bait is fused at its C-terminus to the C-terminal half of ubiquitin Cub the yellow

fluorescent protein (YFP) and a hybrid TF consisting of the E coli DNA binding protein

LexA and the AD of VP16 from the herpes simplex virus collectively known as the CYT

tag (Fig 5) (5 62) The other protein of interest the prey which can be either cytosolic

or membrane bound is fused at its N or C terminus to the N-terminal half of ubiquitin

harbouring an Ile13Gly mutation designated NubG that serves to counteract the natural

affinity Cub and wildtype Nub have for one another These prey protein can either be

specifically selected or consist of entire cDNA or genomic DNA libraries Both bait and

prey proteins are carried on a plasmid and are co-expressed in a Saccharomyces

cerevisiae host cell If the bait and prey proteins interact Cub and NubG are brought into

close proximity and can overcome the counteracting activity of the glycine mutation (Fig

22

5) This results in the reconstitution of a pseudoubiquitin molecule whose subsequent

recognition by cytosolic deubiqutinating enzymes (DUBs) effectively releases the TF

which can freely enter the nucleus and activate the transcription of reporter genes

allowing for growth on selective medium and subsequent verification using an X-gal (5-

bromo-4-chloro-3-indolyl-β-D-galactopyranoside) screen (5)

Though a powerful genetic approach the tMYTH assay was not well suited for

the study of all membrane proteins Overexpression of some membrane proteins could

occur due to the exogenous expression of the bait proteins which would result in self-

activation of the reporter system in the absence of an interaction (32) To overcome this

integrated MYTH (iMYTH) was developed (32) where the bait tag was integrated into

the yeast chromosome providing an endogenous level of expression thereby avoiding

the potential risk of self-activation

Figure 5 Schematic of the iMYTH system (A) A membrane protein of interest the bait shown in blue

is fused to Cub YFP and the TF LexA-VP16 The prey protein shown in pink is fused to NubG The

reporter genes in the nucleus are in the off state (B) If the bait and prey proteins interact pseudoubiquitin

is reconstituted and cleaved at its C-terminal end by DUBs which releases the TF into the nucleus where it

binds to the LexA operator sites (lexA ops) and activates the reporter genes HIS3 ADE2 and lacZ Based

on Paumi et al (5) Iyer et al (67) and Stagljar et al (62)

23

Since its development variations of the MYTH assay have been successfully used

to find interacting partners of the yeast Ycf1p transporter in a large-scale library screen

(32 68) to characterize the interaction between the yeast endoplasmic reticulum (ER)

proteins Msn1p and Rer1p (7) to find novel interactors of the mammalian ErbB3

receptor from human cDNA libraries (69) and even to investigate interactions between

plant sucrose transporters (70) In addition MYTH has been used to find interactors of

Tmem176B and Tmem176A both of which are involved in the maintenance and

maturation of dendritic cells (71) to elucidate binding partners of the human papilloma

virus (HPV) E5 protein and better understand the virus phogenicity (72) and to identify

small GTPases that modulate the surface expression of P-glycoprotein (73) among others

(74-76)

The iMYTH system has several advantages over the traditional Y2H assay the

first being that it is specifically designed for but not limited to the investigation of

interactions between full-length membrane proteins In addition unlike the Y2H system

iMYTH does not require the interaction to occur in the nucleus which allows for the

protein under investigation to undergo post-translational modifications and have proper

localization This system is well suited for the study of many types of integral membrane

proteins irrespective of their localization as long as the Cub-TF and NubG moieties

fused to their respective proteins are located in the cytoplasm and therefore accessible to

DUBs (7 67) This requirement is a disadvantage of the system as it cannot be used to

study transmembrane proteins whose N and C termini are both located outside of the

cytosol or to study proteins which reside within the inner mitochondrial membrane as

DUBs are exclusively found in the cytosol (69)

24

111 Thesis Rationale

Protein-protein interactions play an important role in numerous events that occur within a

cell Nearly one third of a given organismrsquos genome encodes membrane proteins which

due to their hydrophobic nature have proved difficult to study using conventional

methods and as a result interactions involving these proteins are severely

underrepresented in genome-wide screens Considering the implications ABC transporter

proteins have for a diverse set of human diseases and multidrug resistance understanding

their mechanism of action and function is of great importance One of the first steps

towards these goals is the elucidation of complete protein interaction maps or

interactomes which can be effectively done using the iMYTH system The goal of this

work is to generate an interactome for each of the two yeast ABC transporter proteins

Pdr12p and Ste6p using the iMYTH assay As a weak acid anion pump conferring

resistance to food preservatives Pdr12p has implications for food spoilage preservation

and while Ste6p is a mating pheromone transporter it is also a homologue of human P-

glycoprotein which has been implicated in many cancers The identification of novel

protein interactors will allow further characterization of the function of Pdr12p and

Ste6p and identify unknown protein regulators Any knowledge gained from the

interactome of these proteins may lead to the better understanding of their human

homologues and identification of novel drug targets

25

CHAPTER 2

MATERIALS AND METHODS

26

21 Yeast Strains Media and Growth Conditions

Yeast strains used in this study and their relevant genotypes can be found in Appendix I

The media and solutions used for iMYTH screening and throughout this study were

prepared as previously described (67 77) and can also be found in Appendix I

22 Generation of Endogenously CT- and CYT-tagged Bait Proteins

Full length C-terminally tagged Pdr12p and Ste6p baits were generated as previously

described in detail (32) Briefly it involved the PCR amplification of a fragment from

the pCYT-L3 plasmid containing the Cub-YFP-TF (CYT) cassette and the KanMX

resistance marker gene or the L2 plasmid containing the Cub-TF (CT) module This

PCR product was then transformed into the L40 yeast reporter strain and through

homologous recombination integrated into the chromosome resulting in bait strains with

tagged PDR12 and STE6 genes Colony PCR and sequencing were used to verify the

correct orientation of the tag (32)

23 Construction of the Prey Random Genomic DNA and cDNA Libraries

The yeast cDNA library was purchased from a commercial source (Dualsystems Biotech

Switzerland) and prepared as previously described (67) The genomic DNA library was

prepared in house (32) The prey plasmids of both libraries carry the TRP1 marker

24 Verifying Proper Localization of CYT-tagged Bait Proteins

To examine the localization of CYT-tagged Pdr12 and Ste6 proteins the YFP which is

part of the tag was utilized Freshly grown cells were washed prior to being resuspended

in 100 μL of ddH2O Two microlitres of resuspended cells were spotted on a glass slide

and covered with a cover slip Prior to viewing with the YFP filter a drop of cedar wood

immersion oil was spotted on the coverslip The fluorescence was viewed at 503 nm

wavelength with a fluorescence microscope

27

25 NubGNubI Test

This test was performed in order to verify the correct expression and lack of self-

activation of the CT-tagged bait proteins Two unrelated proteins Ost1p an ER

membrane protein and Fur4p a plasma membrane protein are fused to either NubG or

NubI and are used as control plasmids for this test (see Appendix I) The plasmids

pOst1-NubG and pFur4-NubG are used as negative controls while pOst1-NubI and

pFur4-NubI are used as positive controls An overnight culture of Pdr12-CT and Ste6-

CT was grown and the next day was used to inoculate a 10 mL culture at a starting

OD600 = 015 Once cells reached mid-log phase (OD600 = 06) they were pelleted

washed and resuspended in 1 mL of sterile ddH2O For each transformation 100 microL of

resuspended cells 1 microL of positive or negative control plasmid and 300 microL of

Transformation Master Mix (see Appendix I) were combined and mixed The mixture

was then incubated at 30degC with shaking for 30 minutes after which it was heat

shocked at 42degC for 40 minutes The mixture was then pelleted and the cells

resuspended in 09 NaCl and plated on SD-W plates to select for the presence of the

plasmid Plates were incubated at 30degC for 2-3 days After growth a single colony from

each transformation plate was picked and resuspended in 150 microL of sterile ddH2O

(undiluted sample) Four serial 10-fold dilutions were prepared from the undiluted

sample and 5 microL of each dilution was spotted on SD-W plates to verify that the

transformation was successful and on SD-WH to select for the activation of the reporter

gene system Plates were again grown at 30degC for 2-3 days and results were then

assessed

28

26 Verification of C(Y)T-tagged Bait Functionality

261 Generation of Deletion Mutants

Deletion mutants of Pdr12p and Ste6p were generated via homologous recombination

First the Kanamycin resistance (KanMX) and Nourseothricin resistance (Nat) cassettes

from the L2 and p4339 plasmids respectively were PCR amplified using primers

complimentary to the cassettes with over-hang sequence complimentary to the regions

flanking each gene Refer to Appendix II for primer sequences and PCR protocols This

PCR product was then transformed into the L40 yeast strain as follows A 5 mL

overnight culture of L40 was grown and was used the next day to inoculate a 10 mL

culture at a starting OD600 = 015 Once cells reached mid-log phase (OD600 = 06) they

were pelleted washed and resuspended in 1 mL of sterile ddH2O Per reaction 100 microL

of resuspended cells 20 microL of PCR amplified cassette and 300 microL of Transformation

Master Mix (see Appendix I) were combined and mixed well Reactions were then

incubated at 30degC with shaking for 30 minutes after which they were heat shocked at

42degC for 40 minutes The mixture was then pelleted and the cells were resuspended in 4

mL of YPAD and left at 30degC with shaking overnight The cultures were then pelleted

and the cells were resuspended in 09 NaCl and plated on YPAD-Nat or YPAD-G418

plates to select for the presence of the appropriate cassette Plates were incubated at 30degC

for 2-3 days

262 Verifying Deletion Mutants

Deletion mutants were verified by growth on medium containing the appropriate

antibiotic and via PCR on purified genomic DNA A phenolchloroformisoamyl

alcohol-based method was used to extract the genomic DNA as follows A 2 mL

overnight culture of each deletion mutant was grown Cells were pelleted and

29

resuspended in 200 microL of lysis buffer (2 Triton X-100 1 SDS 100 mM NaCl 10

mM Tris-Cl pH=80 1 mM EDTA ddH2O) To this 200 microL each of 05 mm glass beads

and phenolchloroformisoamyl alcohol (25241) were added and the reaction was

vigorously vortexed for 5 minutes The mixture was pelleted and the top layer

transferred to a new tube To this 100 microL of chloroform was added and the mixture was

vortexed for 30 seconds Again 150 microL of the top layer was transferred to a new tube

and 375 microL of 100 EtOH was added The reaction was incubated at -20degC for 30

minutes to allow DNA to precipitate This was then spun down at 14000 rpm for 5

minutes and the pellet was washed in 400 microL of 70 EtOH which was kept at -20degC

Once again this was spun down aspirated and allowed to dry at RT for 5 minutes The

DNA pellet was resuspended in 50 microL of elution buffer For PCR 1microL of this genomic

DNA and primers complimentary to the region outside of the bait genes were used along

with and internal primer for the Nat cassette Refer to Appendix II for primer sequences

and the TaqPfu PCR protocol

263 Verifying Pdr12-C(Y)T Function

To test whether the C(Y)T tag interfered with Pdr12p function as an efflux pump a

Sorbic Acid Stress Assay was performed Colonies of WT PDR12-C(Y)T pdr12Δkan

and pdr12Δnat cells were resuspended in 100 microL of sterile ddH2O (undiluted sample)

From this 3 10-fold serial dilutions were made and 3 microL of each dilution as well as the

undiluted sample were spotted out on YPAD medium and YPAD plates containing 3

mM Sorbic Acid Plates were incubated at 30degC for 2-3 days

264 Verifying Ste6-C(Y)T Function

In order to verify that the C(Y)T tag did not impair the ability of Ste6p to export the

mating pheromone a-factor out of the cell a Mating Assay was performed First a streak

30

of each of the reporter strains BY157 [MATa] and BY158 [MATα] was made vertically

on YPAD medium Intersecting each of these two streaks horizontally were the query

strains BY4743 (aα) BY4741 (a) BY4742 (α) STE6-C(Y)T and ste6Δnat The plate

was incubated at 30degC overnight The next day a thin layer of the intersection of the

reporter and query strains was replica plated on an SD Minimal medium plate and

incubated at 30degC overnight

27 The iMYTH Assay

271 Large Scale Transformation

A detailed protocol on how to perform the iMYTH assay has previously been published

(32 77) Both of the strains expressing the bait proteins Pdr12-CT and Ste6-CT were

transformed with each of the yeast cDNA and genomic DNA libraries using the standard

lithium acetate method (6) Briefly A 50 mL overnight culture of a bait strain was grown

and the next day used to inoculate a 200 mL culture at an OD600 = 015 Once cells

reached mid-log phase (OD600 = 06) they were divided into four 50 mL Falcon tubes

(per 200 mL of culture) pelleted washed in 40 mL of cold sterile ddH2O pelleted again

and resuspended in 1 mL of LiOacTE mix (1 M LiOAc 10X TE pH 75 sterile ddH2O)

This was then transferred to an eppendorf tube pelleted and resuspended in 600 microL of

LiOAcTE mix To each Falcon tube 10 microL of the appropriate library 600 microL of the

resuspended bait cells and 25 mL of Transformation Master Mix (see Appendix I) was

added This was vortexed and incubated in a 30degC waterbath for 45 minutes and mixed

every 15 minutes After incubation to each tube 160 microL of DMSO was added The

reactions were then mixed and heat shocked at 42degC for 20 minutes Cell were then

pelleted resuspended in 3 mL of 2X YPAD and pooled into one Falcon tube The cells

were allowed to recover in the 30degC shacking incubator for 90 minutes Cells were then

31

pelleted resuspended in 49 mL of 09 NaCl solution and plated onto SD-W medium

The plates were incubated at 30degC for 2-5 days

272 Patching and Recovering Putative Interactors

Colony patching was done using the QPix 2 XT robot (Genetix) First colonies of

transformed cells were picked and resuspended in 80 microL of liquid SD-W medium in a

384-well plate format These plates were then incubated at 30degC for 2 days following

which the robot patched the cells onto SD-WH + X-gal plates After two days at 30degC

blue colonies were picked and plated onto SD-W plates and were again grown for 2 days

at 30degC Colonies were then handpicked and placed into a sterile 96-well block

containing 125 mL of liquid SD-W in each well covered with a breathable foil and

grown for 2 days at 30degC with shaking Cells were then pelleted and resuspended in

Lysis Buffer (see Appendix I) and the plates were once again covered with breathable

foil and incubated for 2 hours at 37degC Prey plasmids containing putative interactor

proteins were then recovered from yeast using the Nucleospin Multi-96 Plus Plasmid

miniprep kit following the standard protocol (Macherey-Nagel Germany)

273 Amplification and Recovery of Prey Plasmid DNA

Highly competent XL10 Gold E coli cells were prepared according to the Inoue method

(78) and were used to amplify the prey plasmids obtained from yeast This protocol was

done in a 96-well format E coli cells stored in a 96-well PCR plate were thawed on

ice and to each well containing 100 microL of cells 10 microL of yeast miniprep DNA was

added The reactions were then incubated on ice for 20 minutes heat shocked for 45

seconds at 42degC and incubated on ice for 2 more minutes The reactions were then

transferred to a tube containing 900 microL of LB medium and allowed to recover at 37degC for

an hour Cells were then pelleted half of the supernatant was removed and the cells

32

were resuspended in the remaining half of the LB medium The cells were then plated

onto LB-Amp plates and grown overnight at 37degC The following day single colonies

from each transformation reaction were picked and placed into a sterile 96-well block

containing 12 mL of TB liquid medium (see Appendix I) plus 100 microgmL Ampicillin in

each well The block was incubated for two days at 37degC with shaking Cells were then

pelleted and the prey plasmids were recovered from the E coli using the Nucleospin

Multi-96 Plus Plasmid miniprep kit (Macherey-Nagel Germany) DNA was eluted in a

final volume of 75 microL

274 Prey Identification

Potential interactors were sequenced and identified via a BLAST search using sequence

data from the Saccharomyces Genome Database (SGD) The best hits in frame with the

tag were identified and accepted it if their expect value was no greater than 001

Ubiquitin components of the ribosome and short unidentifiable peptide sequences were

then removed as were any hits encoded in the mitochondria In addition functional

description and localization were used to assess the likelihood of potential candidates

being putative interactors

275 Bait Dependency Test

The bait dependency test was done in order to verify the specificity of the potential

interaction Recovered prey plasmids identified from the iMYTH screens were re-

transformed back into their respective bait strains from which they were originally

identified In parallel these same prey plasmids were transformed into a strain

containing an unrelated artificial bait a protein consisting of the human CD4

transmembrane domain fused to Cub and a MATα signal sequence to direct it to the

membrane The plasmids pOst1-NubG and pFur4-NubG were used as negative controls

33

while pOst1-NubI and pFur4-NubI were used as positive controls (see Appendix I)

Transformations were done in a 96-well plate format using the standard lithium acetate

method (6) A 5 mL overnight culture of each of the Pdr12-CT and Ste6-CT bait strains

as well as the artificial bait strain was grown and the next day used to inoculate a 75 mL

culture at an OD600 = 015 Once cells reached mid-log phase (OD600 = 06) they were

pelleted washed in 40 mL of cold sterile ddH2O pelleted again and resuspended in 375

mL of sterile ddH2O To each well 1microL of prey plasmid 40 microL of the appropriate

resuspended bait cells and 120 microL of transformation master mix (50 PEG 1M LiOAc

2 mgml ssDNA) was added This was mixed using a pipette The plate was then

incubated for 30 minutes at 30degC with shaking The cells were heat shocked at 42degC for

40 minutes pelleted and the supernatant was removed Cells were resuspended in 50 microL

of 09 NaCl and plated onto SD-W plates to select for the presence of the prey plasmid

Plates were incubated at 30degC for 2-4 days Three colonies for each transformation

reaction were picked and resuspended in 100 microL of sterile ddH2O 3 microL of resuspended

cells were plated onto SD-W plates to verify retention of the prey plasmid and ensure

comparable growth between spots and onto SD-WH + X-gal to select for the interaction

of bait and prey

28 Generation of Double Deletion Mutants

Double deletion mutants of Pdr12p and the identified interactors of this protein were

generated through mating and tetrad dissection Single deletions of the genes encoding

the Pdr12p interactors were obtained in the BY4741 [MATa] strain from the yeast

deletion collection (79) where the gene of interest is replaced with the KanMX cassette

These strains were then mated with the DDN1242 (pdr12Δnat) strain generated in this

study (as described in section 261) by intersecting the streaks of each strain on YPAD

34

plates After an overnight incubation at 30degC the intersecting cells were streaked out

onto YPAD + Nat + G418 plates to obtain single colonies with both Kanamycin and

Nourseothricin resistance which would be found in cells that have successfully mated

These plates were incubated at 30degC overnight and the next day a thin layer of cells from

a single colony was streaked out onto Sporulation medium plates These were left for 7-

10 days at RT to form tetrads Following sporulation a small amount of cells was picked

up with a sterile toothpick and placed in a tube containing 50 microL of zymolyase solution

(50 microgml zymolyase 1M sorbitol) to digest the spore ascus of the tetrads The cells

were incubated for 5 minutes at 30˚C after which cells were placed on ice and 800 microL of

sterile ddH2O was added to stop the reaction 20 microL of the cells were spread across a

YPAD plate and the tetrads were dissected with a dissecting microscope These plates

were incubated at 30˚C for 2-4 days After growth each one of the colonies was plated

onto a YPAD + G418 as well as a YPAD + Nat plate to determine which had both of the

drug selection markers Those that were verified as having both resistance cassettes were

genomic prepped (as described in section 262) and verified via PCR The double

deletion strains pdr12Δnat pdr5Δkan pdr12Δnat pdr10Δkan and pdr12Δnat

pdr11Δkan were also verified via sequencing Refer to Appendix II for primer

sequences and the Phusion Master Mix PCR protocol

29 Generating Full-length tagged Pdr5p Pdr10p and Pdr11p

291 Gap Repair Method

To generate full-length versions of the three other ABC transporters identified in the

Pdr12p screen gap repair cloning of the genes into the prey plasmid pPR3N was

performed First the genes encoding Pdr5p Pdr10p and Pdr11p were PCR amplified with

primers that have homology to the plasmid but will result in the exclusion of the NubG

35

module when recombined Refer to Appendix II for primer sequences and the Phusion

Master Mix PCR protocol For the digest of pPR3N 15 microL of plasmid 1 microl of the SfiI

enzyme (Fermentas) 5 microL of Buffer G and 29 microL of ddH2O were combined and

incubated at 50˚C for 3 hours The PCR product and digested plasmid were then

transformed into yeast as follows (80) A 5 mL overnight culture was grown to

saturation For each reaction 250 microL of cells were pelleted and the supernatant was

removed To each tube 80 microL of 50 PEG 10 microL of each of 1M DTT and 2M LiOAc

50 microL of ssDNA 25 microL of the PCR product and 5 microL of digested plasmid were added

This was briefly vortexed to mix and incubated at 45˚C for 30 minutes The reactions

were the vortexed for 1 minute at 10000 rpm the supernatant was removed and the cells

were resuspended in 100 microL of sterile ddH2O The entire volume was plated onto SD-W

plates and grown at 30˚C for 2-3 days A 5 mL overnight culture was grown and

plasmids were extracted using the EZ-10 Spin Column Plasmid DNA Kit (BioBasic) after

the yeast cells were vigorously vortexed with 200 microL of 05 mm glass beads for 10

minutes

292 Gateway Cloning

The genes encoding Pdr5p Pdr10p and Pdr11p were PCR amplified from yeast genomic

DNA using primers that would introduce flanking attB1 and attB2 sites These fragments

were then cloned into the pDONR223 plasmid (Invitrogen see Appendix I) using the BP

Clonase reaction (Invitrogen) following the procedure outlined by the manufacturer

This was then transformed into library efficiency DH5α competent E coli cells as

follows E coli cells were first thawed on ice then 100 microL of cells and 10 microL of the BP

reaction mix were combined The reactions were then incubated on ice for 20 minutes

heat shocked for 45 seconds at 42degC and incubated on ice for 2 more minutes The

36

reactions were then transferred to a tube containing 900 microL of SOC medium and allowed

to recover at 37degC for an hour Cells were then pelleted half of the supernatant was

removed and the cells were resuspended in the remaining half of the LB medium The

cells were then plated onto LB-Spectinomycin plates (see Appendix I) and grown

overnight at 37degC 5 mL overnight cultures of individual colonies were grown up at

37degC in LB-Spectinomycin medium and the plasmids were recovered using the the EZ-

10 Spin Column Plasmid DNA Kit (BioBasic) and standard protocol Each entry clone

was first verified by digestion with BsrGI (Frementas) and then sequenced to verify that

they contained the error free full-length sequence of the gene Once confirmed the LR

Clonase reaction (Invitrogen) was done to sub-clone the entry clones into the pYES-

DEST52 destination vector (see Appendix I) as described by the manufacturer This was

then transformed into E coli as described above and verified by digestion with BsrGI

Finally the pYES-DEST52 vector containing the full-length gene sequence was

transformed into the Pdr12-CYT yeast strain as described in section 291 (80)

210 Functional Assays for Pdr12p

2101 Spot Assays

Spot assays were done on WT single and double deletion strains to determine if any of

the identified interactors of Pdr12p had a role in acid anion efflux Single colonies were

resuspended in 100 microL of sterile ddH2O (undiluted sample) from which up to five 10-

fold serial dilutions were made Three microlitres of the last four dilutions were spotted

onto YPAD plates as well as YPAD plates containing either benzoic or sorbic acid at

concentrations ranging from 1 to 10 mM or the drugs artesunate bortezomib and

rapamycin at various working concentrations Plates were allowed to dry and were then

incubated at 30˚C for up to 7 days but were monitored daily

37

2102 Liquid Panelling Assay

Growth in liquid medium containing either sorbic or benzoic acid was monitored with the

GENios microplate reader (TECAN Switzerland) to evaluate the effect these compounds

had on the double deletion mutants A 96-well plate was divided into two allowing for

two replicates on the same plate Eight query strains consisting of WT single and

double deletions were placed in rows A-H while various concentrations of the sorbic or

benzoic acids were placed in columns 1 through 12 The first column only contained

YPAD Cells were inoculated in all wells at an initial OD600 = 006 and the plates were

then sealed with PE foil and placed in the reader for 200 reads (2 days) at 30˚C The

same approach was taken for the drug haloperidol The data was then graphed using

Microsoft Excel Refer to Appendix I for acid media preparation

2103 Co-Immunoprecipitating Interacting Proteins of Pdr12p

Yeast co-immunoprecipitations were done by modifying a previously published method

(81) as follows A 5 mL overnight culture of the bait strain transformed with the

appropriate prey-expressing plasmid was grown and the next day used to inoculate a

200 mL culture at OD600 = 0001 Cells were grown overnight to be at mid-log phase

(OD600 = 05-06) spun down and resuspended in 150 microL of ice-cold lysis buffer (50 mM

HEPES pH=75 100 mM NaCl 10 (vv) glycerol 1mM EDTA 100 mM PMSF 1 M

DTT 500 mM NaF 100 mM Na-o-vanadate 20 mgmL TLCK 10 mgmL aprotinin and

1 mgmL each of pepstatin A and leupeptin) and kept on ice To this 300 microL of cold 05

mm glass beads was added and the cells were lysed via vortex at 4˚C for 10 minutes

Using a 25G ⅝ needle a hole was made in the bottom of the tube and the lysate was

quickly spun into a new tube To this 300 microL of lysis buffer and 60 microL of detergent

(C12E8 or Triton-X 100) (1 final) was added The lysate was incubated at 4˚C on a

38

nutator for 2 hours After the incubation the samples were clarified by spinning for 15

minutes at 0˚C and 5000 x g 50 microL of the extract was saved as the total cell lysate

(TCL) fraction to which 50 microL of 2X SDS loading buffer was added The remaining

extract was immunoprecipitated with 8 microL of either anti-VP16 (Sigma Oakville ON) or

anti-HA (Santa Cruz Biotechnology Santa Cruz CA) antibody via a 2 hour incubation

on the nutator at 4˚C The samples were then clarified by spinning for 10 min at 0˚C and

5000 x g and transferred to a tube containing 30 microL of Protein G Sepharose beads pre-

washed in lysis buffer This was incubated for an hour on the nutator at 4˚C The beads

were then washed 5 times in 500 microl of lysis buffer by rotating 5 minutes on the nutator at

4˚C and spinning for 1 minute at 4˚C and 5000 x g The beads were then resuspended in

30 microL of 2X SDS loading buffer All samples were stored at -20˚C until ready for SDS-

PAGE analysis

2104 Western Blot Analysis

Proteins were resolved by SDS-PAGE on 8 gels for the bait and 15 gels for the prey

which were run at 110 V for 90 minutes (Bio Rad Mini-PROTEAN Tetra System) This

was transferred to a PVDF membrane which was activated in 100 methanol and rinsed

in ddH2O The transfer apparatus (Bio Rad Criterion Blotter) was run at 300 mA for 90

minutes The membranes were then blocked in 5 milk in 1X TBST (see Appendix I)

for 2 hours at RT washed 3 times for 5 minutes each in 1X TBST and incubated

overnight at 4˚C with rocking in primary antibody (11000) in 1 milk in 1X TBST

Pdr12-CT was detected with an anti-LexA (Santa Cruz Biotechnology Santa Cruz) (see

Appendix I) antibody and the various preys were detected with an anti-HA (Roche) anti

V5 (Invitrogen) or anti-His (Cell Signalling) antibody (see Appendix I) The following

morning membranes were washed 10 times for 5 minutes each in 1X TBST then

39

incubated with secondary antibody (14000) in 01 milk in 1X TBST for 1 hour at RT

with rocking For the bait and full-length prey anti-mouse IgG linked to horseradish

peroxide (GE Healthcare UK) was used as the secondary and anti-rat IgG linked to

horseradish peroxide (Cell Signalling) was used for the truncated preys (see Appendix I)

Membranes were again washed 10 times for 5 minutes each in 1X TBST then incubated

in 5 mL of SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) for 5

minutes with rocking The membrane was then placed between overhead sheets in a

cassette and the films HyBlot CL (Denville Scientific) and AmershamHyperfilm (GE

Healthcare) were developed at various time intervals The strains L40 Pdr12-CT not

transformed with the prey and lysis buffer with the antibody were used as controls

211 Extending Ste6p Duration at the Plasma Membrane

In an attempt to find better screening conditions for Ste6p the yeast mating pheromone

α-factor was used in an effort to accumulate and maintain the protein at the plasma

membrane A 5 mL overnight culture of WT Ste6-CYT and DDS0640 (sac6Δnat)

strain cells was grown in YPAD The next day it was used to inoculate another 5 mL

culture at an OD600 = 015 The cells were grown to an OD600 = 03-04 at which time

various concentrations of α factor were added to the medium and incubated at 30˚C for 1

hour All strains also had an untreated control Cells were pelleted washed with ddH2O

pelleted again and resuspended in 100 microL of ddH2O Two microlitres of resuspended

cells were spotted on a glass slide and covered with a cover slip Prior to viewing with

the YFP filter a drop of cedar wood immersion oil was spotted on the coverslip The

fluorescence was viewed at 503 nm for YFP using a fluorescence microscope

40

CHAPTER 3

RESULTS

41

31 Endogenously CT and CYT-tagged Bait Proteins Successfully Generated Saccharomyces cerevisiae L40 iMYTH reporter strains expressing endogenously CT and

CYT tagged ABC transporter baits were constructed prior to my start in the lab The CT

and CYT cassettes were amplified from the L2 and L3 plasmids respectively and

integrated downstream of and in frame with the PDR12 and STE6 genes via homologous

recombination PCR of genomic DNA and sequencing were used to verify the correct

tagging of the PDR12 and STE6 ORFs

32 CYT-tagged Integrated Bait Proteins Strains Localize Correctly

To verify that the tagged bait proteins localized properly within the cell CYT-tagged

baits were visualized via the yellow fluorescent protein (YFP) within the CYT tag using

fluorescence microscopy prior to my start in the lab Both Pdr12p and Ste6p are

reported to be localized to the plasma membrane (16 41) As can be seen in Fig 6A the

signal from the YFP of Pdr12-CYT is localized to the plasma membrane of the cell

indicating that the CYT tag does not impair the proper localization of this protein In the

case of Ste6-CYT the signal is diffuse throughout the vacuole (Fig 6B) most likely due

to the short half life this protein has at the plasma membrane and its rapid recycling

within the cell (41 43) However this does not indicate that the tagged protein is

improperly localized Both bait proteins appear to localize to their reported compartment

in the presence of the CYT tag and were further validated for functionality and

suitability in iMYTH screening (see below)

42

Figure 6 CYT-tagged bait protein localization The left of each panel is the YFP channel and the right

is the overlay with DIC A) Pdr12-CYT localizes to the plasma membrane B) Ste6-CYT signal is diffuse

within the vacuole which is consistent with previous reports of its rapid endocytosis Scale bar is 4 microm

Snider et al (unpublished data)

33 Tagged Bait Strains Pass NubGNubI Test

The purpose of this test it to verify the proper expression of the integrated bait proteins

once their proper sequence has been confirmed as well as to verify that they are not self-

activating which would result in false positives during iMYTH screening The

NubGNubI test was done prior to my start in the lab by transforming the bait proteins

with control plasmids Fur4-NubI and Fur4-NubG (refer to Appendix I Table 3 for

details) The results of this test indicated that both Pdr12-CT and Ste6-CT are expressed

and not self activating (Fig 7) This is evident by the ability of transformed bait strains

to grow on medium selective for interaction of bait and prey constructs (SD-WH) only in

the presence of the positive control plasmid Fur4-NubI which harbours the WT N-

terminus of ubiquitin which spontaneously interacts with the C-terminus of ubiquitin In

the presence of the Fur4-NubG plasmid which contains the mutated version of N-

terminal ubiquitin and as should not interact with the bait proteins there is no growth on

the selective medium Therefore based on the results obtained both integrated CT-

tagged Pdr12p and Ste6p were deemed suitable for use in iMYTH screening

43

Figure 7 NubGNubI test for integrated bait strains Control prey plasmids used to transform the CT-

tagged integrated bait strains are listed on the left Serial dilutions of transformed colonies were spotted on

to medium selective only for the presence of plasmid (SD-W) to indicate that the transformation was

successful and onto medium selective for interaction (SD-WH) to evaluate the interaction between the bait

and prey A) Pdr12-CT only interacts with Fur4-NubI on selective medium B) Ste6-CT only interacts

with Fur4-NubI on selective medium Growth of strains transformed with NubI controls but not NubG

controls indicates that the bait is being expressed and is not self activating Snider et al (unpublished

data)

34 Functional Analysis of Bait Proteins

341 Pdr12-CT Grows in the Presence of Sorbic Acid

In order to verify that the CT tag did not interfere with the function of Pdr12p as an efflux

pump spot assays on medium containing the commonly used food preservative sorbic

acid were done In the presence of sorbic acid both the WT and Pdr12-CT bait strains

have the same fitness while the deletion mutant strains DDK1240 (pdr12Δkan) and

DDN1240 (pdr12Δnat) are severely impaired in their ability to grow in the presence of

this weak acid (Fig 8) Therefore the CT tag does not affect the function of Pdr12p as

an acid anion efflux pump This assay also served to functionally verify the deletion

strains as the inability to grow on medium containing sorbic acid indicates the successful

deletion of PDR12

44

Figure 8 CT tag does not interfere with Pdr12p function Strains spotted out on to YPAD and YPAD

+ 3 mM sorbic acid medium are listed on the left and the dilution factors are shown above Two individual

colonies for DDK1240 (pdr12Δkan) and DDN1240 (pdr12Δnat) strains were used Pdr12-CT is able to

grow as well as the WT strain on the plate containing the weak acid while the deletion mutants are

compromised in their growth

342 Ste6-CT is Able to Mate

Since Ste6p is involved in the export of the mating pheromone a-factor and therefore

important in the mating of yeast cells a mating assay was performed to investigate what

effect if any the CT tag had on the process After mating on rich medium the cells were

plated onto SD minimal medium (see Appendix I) to examine growth as only cells that

have successfully mated would have the ability to grow on low nutrient medium This is

due to the stress induced by the lack of nutrients in the medium which favours the

formation of haploid spores that are well adapted for survival in unfavourable conditions

for prolonged periods of time and can only be produced by cells that have mated Both

mating control strains BY4741 and BY4742 successfully mated with the opposite

mating type of the reporter strains BY157 [MATa] and BY158 [MATα] as evidenced by

the presence of growth (Fig 9) Ste6-CT strain was also able to mate with the α reporter

strain while the ste6Δnat deletion strain was unable to grow like the diploid control

Therefore STE6 was successfully deleted as determined by the inability of the strain to

45

grow on minimal medium and the CT tag does not impair the export of a-factor out of

the cell as evidenced by growth indicative of mating

Figure 9 Evaluating Ste6-CT function with a mating assay Shown is the replica plate with the mated

intersection plated on SD minimal medium Reporter mating strains a and α were streaked in two columns

while the query strains listed on the left including the diploid and mating controls were streaked

horizontally The diploid by definition cannot mate while BY4741 and BY4742 are used as positive

mating controls Ste6-CT is able to mate while the ste6Δnat deletion strain as expected is not

35 iMYTH Screening Results

351 Large Scale Library Transformation

Both Pdr12-CT and Ste6-CT tagged integrated bait strains were transformed with NubG-

X cDNA (Dualsystems Biotech) and genomic DNA libraries to identify novel interactors

for each Screening was performed until sufficient coverage of each library was

obtained which was considered to be at least two million transformants given that the

complexity of each library is approximately one million clones After multiple rounds of

robotic based screening and selection putative interactors of interest were recovered and

identified via sequencing prior to being used in the bait dependency test The screening

results for Pdr12-CT and Ste6-CT are summarized in Table 1 below The putative

interactors used in the bait dependency test exclude redundant hits ubiquitin components

of the ribosome mitochondrially encoded proteins as well as short unidentifiable

peptides

46



In order to determine which of the putative interactions identified through the large-scale

screen are specific the bait dependency test is performed All potential interactors and

control plasmids were transformed back into their respective bait strains as well as a

strain expressing an artificial bait protein This artificial bait is a synthetic construct

consisting of the human CD4 transmembrane domain fused to Cub and a MATα signal

sequence to direct it to the membrane It is used as the control as it is unrelated to the

bait proteins and is therefore useful for identifying preys which are spurious interactors

possibly binding to components of the CT tag itself or non-specifically to other parts of

the bait Three individual transformant colonies were then selected and plated onto non-

selective and selective media and evaluated for the presence of an interaction As can be

seen in Fig 10 A when transformed with the control plasmids carrying the NubI

constructs Pdr12-CT Ste6-CT and the artificial bait grow on both medium selective for

the presence of the prey plasmid (SD-W) and medium selective for interaction (SD-WH)

However when the NubG version is used growth is seen only on medium selective for

the presence of the prey plasmid as expected Any potential interactor that allows

growth on medium selective for interaction when transformed into the artificial bait

strain is scored as a false positive (Fig 10 B) Thus only interactors that allow growth

47

on medium selective for an interaction when transformed into the original bait strain are

counted as valid hits and used to generate the protein interactomes

Figure 10 An example of a bait dependency test Baits are listed along the top while control plasmids

and various preys are listed on the left side SD-WH + X-gal and SD-WH are media selective for an

interaction SM is used to denote either in panel B SD-W is selective for the presence of prey plasmid and

is used to verify the success of the transformation reaction and ensure comparable growth between spots

(A) Controls used for Pdr12-CT and Ste6-CT Both Pdr12-CT and Ste6-CT display absence of growth on

medium selective for an interaction when transformed with the NubG plasmids indicating they do not self

activate (B) Preys A B and C show false positive hits as in all cases there is growth on medium selective

for an interaction using the control artificial bait strain Preys D and E show an example of a validated hit

for each of Pdr12-CT and Ste6-CT respectively as in both cases there is only growth on medium selective

for an interaction when the prey is transformed into its respective bait

353 Pdr12p Interactome

After the completion of the bait dependency test using all 81 putative interactors detected

in the Pdr12-CT screen 13 were found to be specific These were partially categorized

by their localization according to their description on the Saccharomyces Genome

48

Database and according to gene ontology classification with respect to their biological

process (Fig 11) Notable interactions include three other members of the ABCG

subfamily Pdr5p residues 1150-1268 (EYRAVQSELDWMERELPKKGSITAAEDK

HEFSQSIIYQTKLVSIRLFQQYWRSPDYLWSKFILTIFNQLFIGFTFFKAGTSLQGL

QNQMLAVFMFTVIFNPILQQYLPSFVQQRDLYEA) Pdr10p residues 1206-1325

(REMQKELDWMERELPKRTEGSSNEEQKEFATSTLYQIKLVSYRLFHQYWRTPF

YLWSKFFSTIVSELFIGFTFFKANTSLQGLQNQMLAIFMFTVVFNPILQQYLPLFV

QQRELYEARER) and Pdr11p residues 326-517 (IQSPYYKHWKAITSKTVQECTRK

DVNPDDISPIFSIPLKTQLKTCTVRAFERIIGDRNYLISQFVSVVVQSLVIGSLFYNIP

LTTIGSFSRGSLTFFSILFFTFLSLADMPASFQRQPVVRKHVQLHFYYNWVETLAT

NFFDCCSKFILVVIFTIILYFLAHLQYNAARFFIFLLFLSVYNFCMVSLFALTA)

Please see Appendix III for sequences of all protein found to interact with Pdr12p With

the exception of Gtt1p and Pdr5p whose fragments were found twice in the Pdr12p

screen all other interacting protein fragments were identified once

Pdr12p was also found to interact with fragments of two proteins involved in the

general stress response Sod1p and Zeo1p which are involved in oxidative stress and the

cell integrity pathway respectively and may have a role in the various processes evoked

in the presence of weak acid stress The interactions between Pdr12p and that of the

fragments of Pdr5p (38) and Pdr10p (82) have previously been reported indicating that

11 of the interactions identified with iMYTH are novel for this protein Of these four

proteins are of unknown function These proteins are also of interest as their roles and

function could be characterized in relation to their interaction with Pdr12p With the

exception of the interaction with Pdr5p the interaction data obtained in this study does

49

not overlap with that of the known interactors of Pdr12p identified by PCA by Tarrasov

et al (2008) This is not unusual between high-throughput large-scale studies as a small

overlap was found between two of the first comprehensive genome-wide analyses of PPIs

in yeast (59) A possible explanation for the low overlap observed is that iMYTH and

PCA are two very different techniques Since a library was used to screen for interactors

the entire genome may not have been covered and if it was it is possible that certain

valid interactions may have been excluded in the initial detection steps simply based on

size specifications fed to the robot In addition it should be noted that the interactions

detected with PCA also had low overlap with other genome-wide PPI screens (38)

Please refer to Appendix IV and VII for the results of the bait dependency tests on all

potential interactors and for a description of the proteins that interact with Pdr12p

respectively

Figure 11 Pdr12p Interactome Circles and diamonds represent proteins that interact with Pdr12p

Diamonds also indicate proteins found in the plasma membrane Each colour on the map corresponds to a

specific biological process based on gene ontology classification which can be found in the legend on the

left hand side

50

354 Ste6p Interactome

For Ste6p 16 potential interactors were subjected to the bait dependency test 14 of

which were identified as false positives The two remaining protein interactions with

fragments of Vps9p and a protein of unknown function Ygl081Wp are novel These

were also categorized by biological process according to gene ontology classification to

generate the interactome (Fig 12) Vps9p is a guanine nucleotide exchange factor that is

involved in the transport of vacuolar proteins (83) and may be involved in the shuttling

of Ste6p to and from the plasma membrane however further studies are needed to

investigate the exact nature of this interaction as well as the function of Ygl081Wp

Three independent fragments of Vps9p were present in the Ste6p screen while only one

fragment of Ygl081Wp was identified Please refer to Appendix V for the sequences of

Vps9p and Ygl081Wp Also see Appendix VI and VII for the bait dependency test

results using all potential interactors and for a description of the proteins that interact

with Ste6p respectively

Figure 12 Ste6p Interactome Circles represent proteins that interact with Ste6p Each colour on the

map corresponds to a specific biological process based on gene ontology classification which can be

found in the legend on the right hand side

36 Generation of Double Deletion mutants with pdr12Δnat

Analyzing the observed phenotype of a given double deletion mutant with or without the

presence of certain compounds allows for the study of genetic interactions If the

phenotype of a double deletion mutant has a combined effect not exhibited by either

mutation alone and which differs from that of the WT it suggests that the interacting

51

genes may have related functions Genetic interactions are generally identified as a result

of a second mutation enhancing or suppressing the original mutant phenotype With

respect to the present study if any of the proteins identified as interactors of Pdr12p are

involved in the weak acid stress response it is expected that the double deletion mutants

have phenotypes that differ from that of the pdr12Δ the respective single deletion and

WT strains More specifically if the double deletion mutant is shown to be more

sensitive or resistant to the presence of weak acids than is either single deletion mutant

and WT it may indicated that the interacting protein and Pdr12p have redundant

functions and compensate for one anotherrsquos absence Conversely if the double deletion

mutant phenotype is not worse than either of the single deletions it may indicate that the

two gene products are required for the same process and act in the same pathway or

complex

Double deletion mutants were generated by mating the DDN1242 (pdr12Δnat)

strain made in this study to a BY4741 strain containing a deleted ORF encoding for an

interacting protein which was either generated through PCR amplification and

homologous recombination or found within the yeast deletion collection (79) After

mating sporulation and tetrad dissection the potential double deletion mutants were

verified for the presence of the deletion cassette by growth on medium containing

antibiotics as well as with PCR Out of the possible 13 eight double deletion strains

were successfully generated (Table 2) One of the interacting proteins Tub2p is

essential and therefore could not be deleted while cassette amplification and integration

failure were reasons as to why Cos8p Ylr154C-Gp and Yml133Cp single deletion

mutants could not be generated It is possible that the primers used had secondary

52

structure that interfered with their binding to the DNA which would not yield an

amplified KanMX cassette with flanking regions of ORF homology Also the PCR

conditions and program may have been too stringent and therefore not ideal for the

amplification of resistance marker The ORF encoding Ylr154C-Gp is only 150 bp long

and though it was successfully amplified its small size most likely interfered with the

integration of the KanMX resistance cassette Though the mating and tetrad dissection

was repeated multiple times for the Pdr12p interactor Yck2p none of the spores could be

verified as double deletion mutants despite the fact that the PDR12 and YCK2 genes are

not linked It is possible that the tetrads dissected were not true tetrads but in fact four

cells clustered together and therefore would not have come from the same genetic

background which would explain the uncharacteristic segregation of resistance markers

These could have been the result of unintentional shaking during the digestion of the

ascus which would disrupt the original tetrad as without the ascus each individual spore

is easier to separate


Deletion Strain Double Deletion Strain

Interactor Protein MATa MATα MATaα

Pdr10 pdr10Δkan pdr12Δnat pdr10Δkan pdr12Δnat



Gtt1 gtt1Δkan pdr12Δnat gtt1Δkan pdr12Δnat

Sod1 sod1Δkan pdr12Δnat sod1Δkan pdr12Δnat

Tma7 tma7Δkan pdr12Δnat tma7Δkan pdr12Δnat

Ybr056W ybr056wΔkan pdr12Δnat ybr056wΔkan pdr12Δnat

Zeo1 zeo1Δkan pdr12Δnat zeo1Δkan pdr12Δnat

Yck2 yck2Δkan pdr12Δnat Not a double deletion

Cos8 NA NA Cassette amplification failed

Tub2 NA NA Essential

Ylr154C-G NA NA Cassette integration failed

Yml133C NA NA Cassette amplification failed

53

37 pdr10Δkan pdr12Δnat Mutant Shows Resistance to Weak Acids

371 Spot Assays

All double deletion mutants generated were subjected to weak acid stress by growth on

solid medium containing increasing concentrations of the commonly used food

preservatives sorbic and benzoic acid in order to deduce if any of the interacting proteins

of Pdr12p also had a role in the cellular response to weak acid stress Out of eight

successfully generated double mutants only one showed an interesting phenotype The

pdr10Δkan pdr12Δnat mutant appears to confer resistance to weak acid stress as it is

able to grow on medium containing unusually high concentrations of the acids whereas

the WT and pdr12Δnat strains are significantly impaired in their ability to grow under

such conditions (Fig 13) The same phenotype is observed for the pdr10Δkan strain

which outgrows the WT These results imply that Pdr10p may have a role in the weak

acid stress response and given that Pdr12p and Pdr10p have been shown to interact

physically with iMYTH their physical interaction may be a mechanism by which they

mediate weak acid resistance Though it has recently been proposed that Pdr10p has a

role in the regulation of Pdr12p (82) the exact nature of this regulation is not clear and

detailed follow-up studies have yet to be performed

54

Figure 13 Weak acid stress assay Concentrations of acid are indicated along the bottom SA is sorbic

acid BA is benzoic acid and YPAD is rich medium Shown are increasing dilutions of cells with the strain

indicated by the legend in the top right hand corner WT indicates control strain As concentrations of both

SA and BA are increased the WT and pdr12Δnat strains lose their ability to grow However the

pdr10Δkan strain and the double deletion strain are able to grow on medium containing 7 mM of either

weak acid No growth is observed for any strain at 8 mM

372 TECAN Liquid Growth Assay

In order to further validate the spot assay results the GENios microplate reader (TECAN

Switzerland) was used to monitor the growth of control and double deletion strains in

YPAD liquid medium containing various concentrations of either sorbic or benzoic acid

Over the course of two days the robot measured and recorded the OD600 of the cells

every 15 minutes which was later graphed and analysed producing a growth curve for

each strain analysed This assay is generally more sensitive and produces numerical

reads as data which eliminates inconsistencies and bias that may occur when estimating

the relative amount of growth by eye As can be seen in Fig 14 as the concentration of

sorbic acid is increased the maximum OD600 the cells reach slowly decreases The

pdr12Δnat strain is unable to exit from the prolonged lag phase induced by the presence

of the weak acid when concentrations of 5 mM acid or greater are present in the medium

55

while the other strains though showing slightly increased lag phases are still able to

overcome the weak acid stress and grow at concentrations of 5 and 10 mM Though none

of the strains are able to overcome the 20 mM concentration of sorbic acid in the time

measured it is important to note that the strain with the shortest lag phase and highest

maximum OD600 throughout the experiment is the pdr10Δkan pdr12Δnat mutant In

addition the pdr10Δkan strain shows a mild resistance to the presence of sorbic acid in

the medium which is comparable to that of the WT strain This was rather unexpected as

the pdr10Δ strain outgrew the WT control in the presence of weak acids (Fig 13)

However with respect to the pdr10Δkan pdr12Δnat mutant the results are consistent

with the observations of the spot assays where the same double deletion mutant was able

to grow on medium containing sorbic acid where the WT strain was not and further

indicate a possible role for Pdr10p in the cellular response to weak acid stress This

result also further confirms a genetic interaction for these two proteins in addition to the

physical one elucidated by iMYTH however the mechanism of action and the role

Pdr10p may play in the weak acid response is still unclear and requires further

investigation

56

Figure 14 Sorbic acid liquid growth assay Concentrations of sorbic acid used are indicated in the top

left hand corner of each graph YPAD is rich medium and contains no sorbic acid The legend is found in

the bottom most graph and shows the strains used The general trend observed is that the maximum OD600

obtained by each strain decreases as the concentration of sorbic acid increases which is not unexpected

The pdr12Δnat mutant strain is unable to grow past concentrations of 5 mM while all strains are trapped

in a prolonged lag phase at 20 mM The pdr10Δkan pdr12Δnat mutant outgrows all other strains at

every other concentration even the wildtype suggesting a role for Pdr10p in the weak acid response

When benzoic acid is used in the medium the same trends are observed (Fig 15)

The pdr12Δnat strain is once again in a prolonged lag phase by 5 mM and all strains

have reduced maximum OD600 values as the concentration of benzoic acid increases The

pdr10Δkan pdr12Δnat mutant once again has the highest tolerance for the presence of

this weak acid in the medium and therefore the highest cell density outgrowing the WT

strain In addition the pdr10Δkan strain once again exhibits a mild resistance to this

weak acid but still has growth comparable to that of the WT strain As observed with the

sorbic acid liquid assay no strain is able to overcome the high anion concentration

57

induced by 20 mM of benzoic acid In addition to being almost identical to the results

obtained with the sorbic acid liquid growth assay these results are also consistent with

those obtained from the spot assays with respect to the pdr10Δkan pdr12Δnat mutant

Given that the results of two very different techniques using two commonly employed

weak acid preservatives show that the pdr10Δkan pdr12Δnat mutant is able to grow at

unusually high weak acid concentrations Pdr10p likely plays some role in regulating the

weak acid stress response andor sensing cellular acid anion concentrations which may

affect the activity of Pdr12p andor other unidentified detoxification pumps

Figure 15 Benzoic acid liquid growth assay Concentrations of benzoic acid used are indicated in the

top left hand corner of each graph YPAD is rich medium and contains no benzoic acid The legend is

found in the bottom most graph and shows the strains used The maximum OD600 obtained by each strain

decreases as the concentration of benzoic acid increases as expected The pdr12Δnat mutant strain is

unable to grow past concentrations of 5 mM while all strains are trapped in a prolonged lag phase at 20

mM The pdr10Δkan pdr12Δnat mutant outgrows all other strains at every other concentration even the

wildtype suggesting a role for Pdr10p in the weak acid response

58

38 A Variety of Drugs Have no Affect on the Double Deletion Mutants

381 Spot Assays

Given that the iMYTH screen identified a fragment of Pdr5p as interacting with Pdr12p

a subset of drugs known to have an effect on pdr5Δ strains were chosen to test if Pdr12p

may also play a role in the transport of drugs out of the cell in addition to pumping out

weak acid anions All single and double deletion mutants generated were spotted onto

YPAD medium containing various concentrations of the drugs artesunate bortezomib

and rapamycin Artesunate is often used to treat Malaria in combination with other

drugs rapamycin is a serinethreonine kinase inhibitor used as an antibiotic and

immunosuppressant while bortezomib is a proteasome inhibitor used for treating

relapsed multiple myeloma and mantle cell lymphoma According to the Saccharomyces

Genome Database deletion mutants of PDR5 have reduced resistance to artesunate and

bortezomib but increased resistance to rapamycin Any variation in the previously

reported phenotypes was evaluated in the deletion mutants with an emphasis on the

pdr5Δkan pdr12Δnat and pdr10Δkan pdr12Δnat deletion strains When spotted

onto medium containing rapamycin the pdr5Δkan and pdr10Δkan strains appear to be

more sensitive than either the WT or the pdr12Δnat strains (Fig 16 B) The result for

both the strains is surprising given that the expected observation for the pdr5Δkan

strain was increased resistance and not sensitivity The fact that pdr10Δkan shows

sensitivity may imply a role in drug transport for this protein however further study is

needed to elucidate its function Neither the pdr5Δkan pdr12Δnat or pdr10Δkan

pdr12Δnat double deletion strains showed increased or decreased resistance to the drug

rapamycin Instead both deletion strains showed fitness comparable to the WT and the

pdr12Δnat strains indicating that the observed sensitivity of the pdr5Δ mutant is

59

dependent on the WT PDR12 gene When the drug artesunate is present in the medium

pdr5Δkan strain is sensitive as expected as is the pdr10Δkan strain (Fig 16 C) which

is consistent with what was observed for this strain in the presence of rapamycin further

indicating a possible role in drug transport for Pdr10p All other strains including the

double deletions are comparable in growth to WT in the presence of artesunate (Fig 16

C) Excluding the pdr5Δkan mutant which shows slight sensitivity no deviation from

WT is seen in any of the other strains when bortezomib is present in the medium (Fig 16

D) All results presented here were consistent between repetitions of this assay

Figure 16 Drug sensitivity assay The strains used in each row are indicated by the legend on the left

hand side WT indicates control strain Concentrations and drugs are indicated above each panel (A)

These vertically sectioned panels show the YPAD control plates for each of the rows The bottom panel

corresponds to the YPAD controls of panel D (B) The pdr5Δkan and pdr10Δkan deletion strains are

unexpectedly sensitive to various concentrations of rapamycin however the double delete in both cases

does not appear to be affected by the presence of the drug (C) When artesunate is present in the medium

as expected the pdr5Δkan is sensitive The results for the other strains are the same as observed in (B)

(D) Bortezomib has no effect on any of the strains tested

60


According to the FitDB (84) the antipsychotic drug haloperidol has an effect on single

deletion mutants of PDR12 PDR5 PDR10 and PDR11 It was chosen for this reason to

test the effects if any it had on the double deletion mutants of these genes Drug

sensitivity of the double deletion strains and appropriate controls was assessed using the

GENios microplate reader (TECAN Switzerland) Strains were grown in YPAD liquid

medium containing increasing concentrations of the drug During the span of two days

the OD600 was automatically measured and recorded and this data was subsequently

graphed and analysed As can be seen in Fig 17 as the concentration of haloperidol

increases there is very little change in the growth curve of the strains when compared to

their growth in medium without the drug When concentrations of drug reach 500 uM

twice the concentration used in the FitDB screen the pdr5Δkan and pdr12Δnat strains

have a significantly increased lag time while all the other strains in addition to having a

slightly prolonged lag phase do not reach as high of an OD600 as seen with lower

concentrations of the drug However the double deletion strains of interest are

comparable in fitness to that of the wildtype strain

61

Figure 17 Haloperidol liquid panelling assay Concentrations of the drug haloperidol are indicated in

the top left hand corner of the graphs The legend indicating the strains is found along the top OD600

readings were taken every 15 minutes for a total of 200 reads or 50 hours The data was then plotted and

analysed Up to 250 uM there does not appear to be any effect of the drug on the growth of the strains

The double deletions remain unaffected at 500 uM while the pdr5Δkan and pdr12Δnat strains have a

prolonged lag phase

39 Increasing Ste6p Duration at the Plasma Membrane

391 Treatment with α-factor

Though the iMYTH screen for Ste6p had sufficient coverage for the library complexity a

relatively low number of potential interactors were identified which was further reduced

to only two actual hits after the bait dependency test Given that Ste6p has a very short

half-life it is possible that it does not exist at the plasma membrane in sufficient levels or

for sufficient duration under standard labarotory growth conditions to allow for the

detection of interactions with the iMYTH assay In order to improve the screening

results of Ste6p conditions that would prolong its stay at the PM and therefore the time

62

it has to interact with other proteins were sought after As the mating pheromone a-

factor exporter which becomes active during mating it was thought the presence of α-

factor might increase the duration and level of Ste6p at the membrane as this would

mimic mating conditions Cells of the Ste6-CYT and the WT strains were left untreated

or were treated with various concentrations of α-factor prior to viewing under the

fluorescence microscope As the concentration of α-factor increases the signal strength

of Ste6p also increases but becomes saturated at 050 microM of α-factor (Fig 18) Though

the signal is stronger implying more Ste6p is present it is completely vacuolar

indicating that it is still being rapidly recycled within the cell and still resides only

briefly at the membrane

Figure 18 Ste6-CYT treatment with α-factor Concentrations of α-factor used are indicated on the left

YFP is the yellow-fluorescent protein channel and Overlay is the YFP channel with DIC Cells were

treated with α-factor for half an hour before being viewed under the microscope As the concentration of α-

factor increases the signal strength of Ste6p increases saturating at 050 microM It is clear the protein is

found exclusively in the vacuole and not at the PM The L40 wildtype strain does not have a YFP tag and

therefore does not exhibit any fluorescence Scale bar is 4 microm

63

3102 Deletion of SAC6

Various methods have been employed to study the trafficking and degradation pathway

that Ste6p follows and this includes blocking the ubiquitination of the protein as well as

studying the effects endocytosis mutants have on Ste6p localization (43) Abolishing the

endocytosis step through the deletion of genes responsible for the process results in the

localization of Ste6p at the membrane When mutated both END4 and SAC6 result in

cells that have defective endocytosis (43) but unlike END4 SAC6 is a non-essential

gene and for this reason was chosen to be deleted in the Ste6-CYT strain This sac6Δ

mutant strain and the WT strain were either left untreated or treated with 050 microM α-

factor to investigate the localization of Ste6p There does not appear to be any difference

between treated and untreated deletion strain cells with respect to signal strength

however the signal does not appear to be clearly localized to one compartment (Fig 19)

In both the untreated and α-factor treated sac6Δ mutant cells there appears to be a subset

of cells exhibiting vacuolar signal and a subset exhibiting possible membrane signal

Unlike the uniform vacuolar signal obtained from treating the Ste6-CYT strain with α-

factor these results hint at an underlying issue such as tag cleavage or tag interference of

the endocytic pathway due to the deletion of SAC6 which may be impairing the proper

localization of this protein

64

Figure 19 Ste6-CYT sac6Δnat localization Strains are indicated on the left hand side while the

untreated and treated cells are shown along the top YFP is the yellow-fluorescent protein channel and

Overlay is the YFP channel with DIC Cells were treated with 050 microM α-factor for 30 minutes before

viewing under the microscope Signal strength between treated and untreated cells is comparable The

deletion mutants exhibit uneven localization as a population (bottom two rows) with cells displaying both

vacuolar (middle panels) and possible membrane (bottom panels) signal being observed Scale bar is 4 microm

65

CHAPTER 4

DISCUSSION

66

41 GO Analysis

Gene Ontology (GO) is used to analyze large data sets such as those obtained from high-

throughput studies for enrichment After the completion of the bait dependency test the

list of interactors obtained for Pdr12p was analyzed for possible enrichment of processes

functions andor common compartments While no significant enrichment was observed

it must be noted that the dataset is relatively small

42 Protein Interactions of Interest

421 iMYTH Identifies an Interaction Between Pdr12p and Pdr5p

The PDR5 gene encodes one of the best characterized ABC transporter proteins Pdr5p

This plasma membrane protein is a powerful pleiotropic drug pump whose

overexpression leads to resistance to cycloheximide and many other drugs (19) while

cells lacking the functional gene product exhibit hypersensitivity to many substrates (11)

This 160 kDa protein also shares similar mechanisms of substrate recognition and

transport with the human MDR1 P-glycoprotein (22) has a large pH tolerance (85) and is

one of the most abundant drug pumps in Saccharomyces cerevisiae (10) In addition to

being members of the same family Pdr5p and Pdr12p have the same reverse topology

consisting of two NBD and two MSD with the NBD preceding the MSD which differs

from the typical ABC transporter topology where the NBD follows the MSD

Mapping protein interaction networks allows for the understanding of the cellular

roles a protein may have as the biological function of a particular protein of interest may

be predicted through the function of an identified interacting partner(s) The

identification of the interaction between Pdr12p and a Pdr5p fragment raises some

interesting questions about the known functions of these two proteins Though Pdr5p has

been classified as a drug pump and numerous studies have demonstrated the broad range

67

of drug substrates it is able to identify and transport the protein may have a role in a

general stress response including weak acid induced stress or perhaps may be more

directly involved in the actual export of the acid anions from the cell as it has been show

with iMYTH to interact with the acid anion pump Pdr12p Conversely identified as a

weak acid anion pump Pdr12p may have an as of yet unknown function in drug

transport Four drugs previously reported to have an effect on Pdr5p were used to

investigate the possible drug transport role of Pdr12p by evaluating double deletion

mutants Though the results obtained here do not provide evidence of Pdr12p

involvement in drug transport (Fig 16 and Fig 17) it must be noted that the four

compounds used represent only a fraction of those known to be transported by Pdr5p In

addition Pdr12p only transports monocarboxylic acids with chain lengths of up to C7

(86) which could imply that any drug transport activity exhibited by this protein would

be more specific than that observed in Pdr5p Interestingly in a study presenting the first

three-dimensional reconstruction of Pdr5p it was reported that upon detergent removal

Pdr5p formed dimers possibly through an interaction between the first cytosolic loops of

two neighbouring Pdr5p molecules (22) This phenomenon has been proposed for other

ABC proteins as well (22) and though it may not be clear whether or not Pdr5p forms

dimers at this time the possibility of it doing so and perhaps forming heterodimers with

other proteins such as Pdr12p cannot be excluded However the biological significance

of this interaction and the means by which it occurs requires further investigation This

may include identifying specific regions of the proteins required for the interaction to

occur by using truncated or mutant forms of both bait and prey proteins as well as

68

biochemically measuring whether or not the rate of transport of certain substrates is

affected by the presence or lack thereof one of the interaction partners


Like Pdr12p Pdr10p is also a member of the ABCG subfamily of yeast ABC transporter

proteins and localizes to the membrane (11) This 1564 amino acid protein is a full-

length transporter regulated by Pdr1p and Pdr3p through cis-acting sites known as PDR

responsive elements (PDREs) (87) Since it is regulated by the same proteins as Pdr5p

and shares more than 65 primary sequence identity to Pdr5p (87) it is thought that

Pdr10p is also a drug pump however the substrates it transports and its actual function

within the cell remain largely unknown Deletion mutants of PDR10 were screened for

sensitivity with four drugs transported by Pdr5p Though the pdr10Δkan strain showed

increased sensitivity to rapamycin and artesunate when compared to WT (Fig 16) no

effect was caused by the drugs bortezomib or haloperidol both of which compromised

the growth of the pdr5Δkan strain (Fig 16 and Fig 17) There still remains a

possibility that Pdr10p is a drug pump like Pdr5p however data obtained in this study

also suggest a completely different role for the protein In addition to the potential role in

drug transport suggested by the drug sensitivity assays a potential role in response to

weak acid stress is also supported by the obtained data and may be the first

characterization of function for Pdr10p The involvement of Pdr10p in the weak acid

response is supported by the observation that cells deleted for both PDR12 and PDR10

exhibit an increased resistance as compared to the wildtype to weak acids such as

sorbic and benzoic (Fig 13 ndash Fig 15) substrates know to be transported by Pdr12p (11)

as well as the observation that Pdr10p is strongly induced by stress conditions (10) The

69

possible mechanisms of action in support of this interaction will be discussed in detail

below

423 iMYTH Identifies Pdr11p as a Novel Interactor of Pdr12p

Unesterified sterol is an essential component of all eukaryotic membranes as it affects

membrane fluidity as well as the activity and localization of many proteins (88) Under

conditions of aerobic growth sterol biosynthesis in yeast is compromised and therefore

sterol uptake is required for cell viability A close homolog of Pdr5p (19) Pdr11p has

been identified as an important mediator of sterol uptake (88) PDR11 encodes a 1411

amino acid full-length ABC transporter protein (11) believed to localize to the plasma

membrane Aside from the involvement in sterol uptake no other information about the

function or substrate specificity is available for Pdr11p The present study was unable to

provide further insight into the function of this protein Though both single and double

deletions of PDR11 were subjected to various conditions including weak acids (data not

shown) and the drug haloperidol (Fig 17) they did not exhibit a phenotype that varied at

all from the WT These results do not provide evidence of a possible role for Pdr11p in

weak acid anion or drug transport however it must be noted that numerous drugs exist

and only a small fraction of them have been examined in the present study and as such

firm conclusions cannot be drawn Given that Pdr12p was shown to interact with a

Pdr11p fragment Pdr12p may also be involved in the uptake of sterol from the external

environment and the two proteins may function together to carry out this process In

addition it is possible that both Pdr12p and Pdr11p have an unknown function that is not

related to either drug or weak acid transport It is clear that to resolve the mystery of

Pdr11p function and the nature of its interaction with Pdr12p further investigation is

needed

70

424 Vps9p is a Novel Interactor of Ste6p

Vps9p was identified through complementation studies of the vacuolar protein sorting

(vps) mutants that missort and secrete vacuolar hydrolases where it was shown to be a

guanine nucleotide exchange factor for the rab GTPase Vps21Rab5 (83 89) The

vacuole of Saccharomyces cerevisiae is an acidic organelle that contains large amounts of

degradative enzymes and is analogous to the lysosome found in animal cells (89)

Vesicle-mediated protein transport a process highly conserved from yeast to higher

eukaryotes and which involves complex cellular machinery plays an important role in

the localization of proteins to the yeast vacuole (83) However the underlying

mechanism involved in the transport of proteins to the vacuole and the vacuolar

membrane remains elusive (89) It has recently been shown that like several other

plasma membrane proteins Ste6p follows the general PURE pathway for its

internalization and that it is ultimately degraded in the vacuole however the trafficking

of the protein to the vacuole is poorly understood (41) It is possible that Ste6p has a

vacuolar targeting signal that is recognized by a vesicle receptor protein such as Pep12p

which would bind Ste6p and initiate its transport into the vacuole via a transport vesicle

Members of the rab GTPase family such as Vps21p are known to be found on transport

vesicles (89) and as such it is not unlikely that Vps9p may bind both the receptor

protein Pep12p bound to Ste6p and the GTPase Vps21p bridging their interaction

which could result in the fusion of the vesicle with Ste6p inside it The vesicle is then

brought to the vacuole where the protein is degraded It is clear that this process is highly

choreographed and may involve a large number of players many of which are still

unknown but the interaction between Ste6p and a fragment of Vps9p may be the starting

71

point in dissecting and gaining an understanding into one portion of a highly complex

signalling pathway

43 Poor Detection of Ste6p Interactions

Although sufficient coverage for the library complexity was obtained in the screens for

Ste6p upon evaluation of the sequenced prey proteins only a small number proved to

contain a potential protein of interest as opposed to a variety of spurious sequences such

as small peptides mitochondrially or ribosomally encoded proteins or empty prey

plasmids In an attempt to increase the number of potential interactors an additional set

of screens was performed However upon the completion of the bait dependency test

only two true interactors remained (Fig 12) It is unlikely that the poor detection of

interacting partners for this protein is due to the inability of the iMYTH assay to detect

these interactions rather it is the nature of Ste6p that complicates the detection of the

proteins it interacts with Ste6p resides only briefly at the membrane with an estimated

half life of 15 ndash 20 minutes and is rapidly recycled (41 43) which may lead to protein

levels at the PM that are too low for the detection of interactions using iMYTH In

addition as the mating pheromone a-factor transporter it is conceivable that Ste6p is

only expressed at higher levels during conditions that would require its localization at the

membrane such as mating between cells In order to find conditions that would stabilize

Ste6p at the membrane two options were explored First it was thought that the

presence of the mating pheromone α-factor would prolong Ste6p retention at the

membrane To this effect cells were treated with various concentrations of α-factor for a

period of time prior to viewing under the microscope Though a clear increase of signal

can be observed Ste6p remains localized to the vacuole indicating that its rate of

turnover was not affected by the presence of α-factor rather it served to induce the levels

72

of Ste6p present in the cell (Fig 18) It has been shown that any mutations that block the

efficient trafficking of Ste6p to the vacuole such as those that affect the secretory

pathway (sec1 sec6 and sec23) or endocytosis (end3 end4 and sac6) result in the

stabilization of Ste6p at the plasma membrane (43) Therefore a mutant with defective

endocytosis was generated to localize Ste6p to the membrane for an extended period of

time Deletion of the non-essential gene SAC6 in the Ste6-CYT strain did not produce

the expected results (Fig 19) YFP signal should only have been observed in the plasma

membrane of the cells viewed However there is still some vacuolar signal and though

there are cells that appear to have plasma membrane localization of Ste6p it could also

be vacuolar membrane localization as in this particular cell the vacuole is almost the

size of the whole cell If in fact the observed membrane localization is vacuolar

membrane it could be due to the ineffective or partial recycling of Ste6p in the sac6

deletion mutant where the disruption of the gene most likely affected parts of the

internalization and trafficking pathway It is also possible that the inconsistency of Ste6p

localization in the cells as a population is due to the cleavage of the CYT tag which

would explain the variant signal patterns observed Though the CYT tag has previously

been shown not to affect Ste6p function (Fig 9) and therefore its proper localization to

the plasma membrane it is possible that in the sac6 deletion strain the tag interferes with

the proper localization of the protein which could result in the strange pattern observed

Neither of the two options explored resulted in the stabilization of Ste6p at the plasma

membrane and as such additional screens were not performed

44 Putative Role for Pdr10p in the Weak Acid Response

The substrates Pdr10p transports remain largely elusive and while it is hypothesized to

be a drug pump the drug assays performed in this study do not support the theory as the

73

four drugs tested here aside from rapamycin and artesunate did not have a significant

effect on PDR10 deletion mutants when compared to WT (Fig 16 and Fig 17)

Surprisingly when testing the effects weak acid stress had on interactors of Pdr12p an

interesting phenotype for the pdr10Δkan pdr12Δnat mutant was observed It has been

shown in this study as well as others (24 34 36) that the deletion of PDR12 results in

cells that are hypersensitive to the presence of weak acids (Fig 8 and Fig 13 ndash 15) A

recently published study has also reported the resistance of their pdr10Δ strain to weak

acids (82) At times in our study the pdr10Δkan strain slightly outperforms the WT

with respect to growth as is evident in the spot assays however it typically performs at

the level of the WT strain when exposed to weak acid medium (Fig 13 ndash Fig 15) Based

on the results of the present work it is unlikely that the deletion of PDR10 results in

resistance to weak acids as no significant difference between the deletion and WT strains

can be observed in liquid growth assays Rockwell et al also concluded that Pdr10p

plays a role in maintaining the proper distribution and function of other membrane

proteins mainly Pdr12p and to perform this function Pdr10p requires Pdr5p Pdr12p and

Lem3p (82) Though not showing a physical interaction between Pdr10p and Pdr12p the

authors do suggest that these two proteins are involved in the weak acid stress response

and somehow work together Contrary to Rockwell et al upon the deletion of both

PDR12 and PDR10 in the same strain weak acid resistance is obtained (Fig 13 ndash Fig

15) further supporting the possibility of Pdr10p as having a role in the weak acid

response How these two proteins mediate weak acid response requires further

investigation but a possible mechanism of adaptation is the upregulation of another as of

yet unknown ABC transporter protein This has been shown to occur for the major drug

74

pumps Pdr5p Snq2p and Yor1p where an increase in resistance to Pdr5p specific

substrates was observed upon the deletion of YOR1 and SNQ2 Likewise the deletion of

PDR5 led to the increased resistance of Snq2p and Yor1p specific substrates (90) If in

fact the deletion of PDR12 and PDR10 results in the upregulation of another ABC

protein a likely candidate is Pdr15p In contrast to its closest homologue Pdr5p Pdr15p

is induced by general stress conditions such as starvation and low pH (10) the latter of

which would be caused by weak acids in the medium In fact it has been shown that

cells deleted for PDR15 exhibit resistance to sorbate (82) which could be the result of

Pdr12p upregulation further supporting the possibility of Pdr15p upregulation for the

acquired resistance in pdr10Δ pdr12Δ cells which is dependent on the deletion of

PDR10 In this model the deletion of PDR10 and PDR12 would initiate a cellular

response that would result in the upregulation of Pdr15p to compensate for the lack of

Pdr12p function resulting in resistance to weak acids Similarly the lack of PDR15

would result in the upregulation of Pdr12p which would be perceived as increased

resistance to weak acids It is possible that Pdr12p and Pdr15p have overlapping

functions with respect to coping with cell stress and therefore Pdr12p Pdr10p and

Pdr15p may function together to mediate weak acid resistance through a mechanism

similar to that of Pdr5p Snq2p and Yor1p upregulation

45 Lack of Expression of Prey Proteins

Co-Immunoprecipitation (Co-IP) experiments are frequently used to confirm and further

investigate PPIs identified through iMYTH The plasmids carrying the fragments of the

proteins Pdr5p Pdr10p and Pdr11p which were pulled out of library screens contained

an HA tag fused to the NubG for detection Though various antibodies concentrations

and conditions were tested the expression of a prey protein could not be detected (data

75

not show) It is possible that a single HA tag is not detectible regardless of the antibody

concentration used or perhaps it is not in a conformation that would allow accessibility

to the antibody A single HA tag has been previously used to show an interaction

between Ycf1p and Tus1p (32) however unlike the three prey proteins of interest in this

study that are plasma membrane bound Tus1p is a cytosolic protein which could

account for its detection with a single HA tag

To produce full-length versions of Pdr5p Pdr10p and Pdr11p gap repair was first

attempted A clone could not be generated as the proteins proved to be toxic as can

happen when membrane proteins are expressed in E coli (54) Gateway cloning was

attempted next however it proved to have limited success as a full-length Pdr5p was

generated though multiple attempts to acquire a clone for Pdr10p and Pdr11p were

unsuccessful The Gateway destination vector carries the V5 and 6XHis epitopes

believed to be easier to detect Once again though the expression of the bait protein

Pdr12p was confirmed the expression of the full-length prey Pdr5p could not be

detected

Considering that the expression of the tagged prey protein in either the truncated

or full-length form could not be detected Co-IP experiments were not done

46 iMYTH as a System for the Detection of PPIs

Large scale iMYTH screens were successfully used to identify novel interactors for the

plasma membrane proteins Pdr12p and Ste6p as well as to detect two previously reported

interactions of Pdr12p This system allows for the sensitive detection of both stable and

transient protein interactions and has successfully been used to explore interactions

between proteins from a variety of organisms using yeast as a host The selection of

putative interactor proteins within this system is a rigorous process that removes frequent

76

flier hits common to cDNA libraries as well as addresses the high false positive numbers

observed in other Y2H technologies This stringency is obtained with the bait

dependency test using an artificially made protein localized to the membrane Though

Pdr12p initially had 81 potential interactor proteins only 13 were identified as true

interactions upon the completion of the bait dependency test thereby removing a large

number of false positive hits The requirement of both growth and blue colour for a true

interaction is just another quality control step in this test In addition identified

interactions can easily be re-confirmed simply by transforming the identified prey back

into the bait strain The major advantages and disadvantages of iMYTH have been

discussed above and while it is an excellent system for the study of membrane proteins

in yeast the continued development and modifications of such systems will be key in

experimental research and could be applied in drug discovery elucidating signalling

pathways and studying viral and host protein interactions

77

CHAPTER 5

FUTURE DIRECTIONS AND CONCLUSIONS

78

51 Concluding Remarks and Future Directions

It was the goal of this study to investigate the interactome of the Saccharomyces

cerevisiae ABC transporter proteins Pdr12p and Ste6p in order to gain insight into their

biological relevance and function The iMYTH assay was used to identify 13 interactions

for Pdr12p two of which were previously reported and two novel interactions for Ste6p

The interactome of Pdr12p has three other ABC transporter proteins which are also

members of the ABCG subfamily as well as several uncharacterized ORFs

Notable identified interactions for Pdr12p include the plasma membrane proteins

Pdr11p Pdr10p and Pdr5p the latter of which is a major drug efflux pump All three of

those proteins have diverse roles ranging from sterol uptake in the case of Pdr11p to drug

transport for Pdr5p Though hypothesized to be a drug pump as well the functional

analyses which focused on the Pdr12p identified interactors indicate a possible role for

Pdr10p in the cellular weak acid response This is supported by the observed resistance

to weak acids in the medium when both PDR12 and PDR10 are deleted This could be

the first characterization of Pdr10p function as well as the potential substrates it may

transport In addition the possibility of Pdr12p and Pdr10p forming a heterodimer

cannot be dismissed as it was shown via iMYTH that these proteins physically interact

Through this physical interaction Pdr10p may regulate the activity of Pdr12p and

perhaps other as of yet unidentified cellular detoxification pumps Though an

interaction with Pdr5p was also identified the data presented here do not support a role

for Pdr12p in drug transport with respect to Pdr5p specific substrates The interaction

with Pdr11p requires further exploration as Pdr12p may have a possible role in sterol

uptake through its association with Pdr11p which would also be a novel role for the

weak acid efflux pump

79

In the case of Ste6p both interactions identified have not been previously

reported and given that one of these is a protein of uncharacterized function further

studies based on Ste6p function could provide insight into the function of Ygl081Wp

The interaction with Vps9p is both interesting and puzzling and while the nature of their

interaction remains elusive it may provide insight into the complex machinery of protein

shuttling and delivery to the vacuole for degradation In the case of Ste6p it was also an

aim to improve the yield of protein interactors identified through iMYTH screening and

to this end both α-factor and the deletion of SAC6 a gene involved in endocytosis were

methods employed in order to stabilize Ste6p at the plasma membrane However neither

method provided the expected result

Given the interesting interactors identified for Pdr12p specifically Pdr5p Pdr10p

and Pdr11p it is of great interest to investigate the nature of their interactions further

The confirmation and characterization of the identified PPIs is a logical first step As the

expression of the identified prey proteins could not be confirmed Co-IP experiments

could not be used to confirm the interaction of Pdr12p with each of Pdr5p Pdr10p and

Pdr11p Along the same lines all the other identified interactions can be further

confirmed in the same manner To show the relevance of an interaction between two

proteins it is useful to try and validate interactions using full-length proteins in the Co-IP

experiments keeping in mind the problems sometimes associated with masking of the

binding sites Though a full-length Pdr5p was successfully generated a clone could not

be obtained for Pdr10p and Pdr11p Therefore the generation of full-length proteins will

be an integral part of confirming these interactions

80

Pdr10p is largely uncharacterized with respect to function as are the substrates it

transports The fact that the pdr10Δ pdr12Δ deletion mutant exhibited resistance to high

concentrations of weak acids present in the medium is a puzzling yet interesting result

one which warrants further investigation Firstly conditions that would yield consistent

and repeatable results should be identified as there is an observed difference between the

performance of the pdr10Δ deletion mutant in the presence of weak acids when grown on

solid and in liquid media It would also be interesting to do co-localization experiments

with Pdr12p and Pdr10p to evaluate their proximity and determine whether or not the

two proteins form a heterodimer to export acid anions form the cell In addition the role

of Pdr15p in the weak acid response should be investigated If in fact this protein is

upregulated upon the deletion of PDR12 and PDR10 measuring the amount of mRNA

present in the cell with and without the weak acid stress would provide some insight into

whether or not this is the protein responsible for the observed resistance to weak acids It

would also be interesting to investigate the effects the deletion of PDR15 by itself or in

combination with PDR12 and PDR10 would have on the cells ability to adapt to the

presence of weak acids in the medium

Although the Pdr5p Pdr10p and Pdr11p identified as interactors of Pdr12p are

truncated forms of the proteins the region involved in the interaction can be further

narrowed down with mutant and further truncated versions of the proteins using the

identified sequence as a starting point In addition the region of Pdr12p required for the

interaction can be determined using the same methods As all of these proteins are

involved in the transport of substrates their interactions can be further investigated by

biochemically measuring the rate of transport The ATPase activity of each transporter

81

protein under different conditions with or without an interacting partner deleted can be

determined by using radioactively labelled substrates or fluorescent dyes

Further investigation is also required to identify the nature of the interaction

between Ste6p and Vps9p the latter of which may have a role in the shuttling of Ste6p to

the vacuole for degradation As mentioned above this process is complex and has many

branches and proteins involved therefore the first step in characterizing this interaction

would be mutational analyses It would be worthwhile to investigate the localization and

degradation of Ste6p in a VPS9 deletion background as well as in strains deleted for

other proteins involved in the same pathway as Vps9p

Given the low number of hits obtained for Ste6p it is clear that the standard

screening conditions of iMYTH need to be adjusted to improve the potential results for

this protein The deletion of SAC6 and the presence of α-factor did not result in the

stabilization of Ste6p at the plasma membrane Given that the screen for this a-factor

transporter yielded only two interactors it would be of interest to identify screening

conditions better suited for this protein andor strains that have Ste6p stabilized at the

membrane as they may lead to the discovery of other interactors of this protein A

possible mechanism would be to employ the use of the end4ts mutant strain identified

through random mutagenesis and shown to be defective in endocytosis (91) The region

containing the mutation could be PCR amplified and introduced into the Ste6-CYT strain

via homologous recombination and once all requirements for iMYTH have been met

this strain could be used to screen for additional interactors of Ste6p Conversely Ste6p

could be CT tagged in the end4ts mutant strain and used in screening

82

As a more general view at the next step creating double deletion mutants of all

the protein interactions identified in this study would allow for further characterization of

the nature of these interactions As well through mutational analysis and functional

assays such as drug or weak acid assays proteins of unknown function identified in the

Pdr12p screen could be characterized The same could be done for the uncharacterized

ORF identified in the Ste6p screen If certain ORFs prove to be essential or problematic

decreased abundance by mRNA perturbance (DaMP) alleles can be made By disrupting

the 3rsquo UTR of a gene either through the introduction of a resistance marker or deletion

these alleles provide a decreased yield of mRNA and therefore gene product In

addition more drugs should be tested in either spot assay or TECAN format to

investigate the possibility that Pdr12p has a role in drug transport like its interacting

protein Pdr5p

Give the prevalence of ABC transporter proteins across species and the fact that

their core domain is highly conserved it is clear that this family of proteins is of

significant importance As such they have been the focus of study for many years which

collectively has yielded a vast amount of knowledge about these proteins and their

function However there is still a substantial amount that can be learned about the

proteins they interact with through which domains this interaction occurs and for some

their function By employing the iMYTH assay in the search for interacting proteins of

yeast ABC transporters a subset of unique interactions for Pdr12p and Ste6p have been

discovered which in combination with functional studies will lead to further

understanding of their biological function In addition through the study of yeast

proteins knowledge and insight can be gained into the function of mammalian

83

homologues which will aid in the further understanding of ABC transporter related

diseases and the discovery of new therapeutics for their treatment

84

REFERENCES

1 Rees D C Johnson E and Lewinson O (2009) ABC transporters the power

to change Nat Rev Mol Cell Biol 10 218-227

2 Higgins C F (1992) ABC transporters from microorganisms to man Annu Rev

Cell Biol 8 67-113

3 Jungwirth H and Kuchler K (2006) Yeast ABC transporters-- a tale of sex

stress drugs and aging FEBS Lett 580 1131-1138

4 Schmitt L and Tampe R (2002) Structure and mechanism of ABC transporters

Curr Opin Struct Biol 12 754-760

5 Paumi C M Chuk M Snider J Stagljar I and Michaelis S (2009) ABC

transporters in Saccharomyces cerevisiae and their interactors new technology

advances the biology of the ABCC (MRP) subfamily Microbiol Mol Biol Rev 73

577-593

6 Gietz R D and Woods R A (2002) Transformation of yeast by lithium

acetatesingle-stranded carrier DNApolyethylene glycol method Methods

Enzymol 350 87-96

7 Stagljar I and Fields S (2002) Analysis of membrane protein interactions using

yeast-based technologies Trends Biochem Sci 27 559-563

8 Decottignies A and Goffeau A (1997) Complete inventory of the yeast ABC

proteins Nat Genet 15 137-145

9 Casal M Paiva S Queiros O and Soares-Silva I (2008) Transport of

carboxylic acids in yeasts FEMS Microbiol Rev 32 974-994

10 Ernst R Klemm R Schmitt L and Kuchler K (2005) Yeast ATP-binding

cassette transporters cellular cleaning pumps Methods Enzymol 400 460-484

11 Bauer B E Wolfger H and Kuchler K (1999) Inventory and function of yeast

ABC proteins about sex stress pleiotropic drug and heavy metal resistance

Biochim Biophys Acta 1461 217-236

12 Tarr P T Tarling E J Bojanic D D Edwards P A and Baldan A (2009)

Emerging new paradigms for ABCG transporters Biochim Biophys Acta 1791

584-593

13 Schmitz G Langmann T and Heimerl S (2001) Role of ABCG1 and other

ABCG family members in lipid metabolism J Lipid Res 42 1513-1520

14 Kusuhara H and Sugiyama Y (2007) ATP-binding cassette subfamily G

(ABCG family) Pflugers Arch 453 735-744

15 Miyahara K Mizunuma M Hirata D Tsuchiya E and Miyakawa T (1996)

The involvement of the Saccharomyces cerevisiae multidrug resistance

transporters Pdr5p and Snq2p in cation resistance FEBS Lett 399 317-320

16 Hatzixanthis K Mollapour M Seymour I Bauer B E Krapf G Schuller

C Kuchler K and Piper P W (2003) Moderately lipophilic carboxylate

compounds are the selective inducers of the Saccharomyces cerevisiae Pdr12p

ATP-binding cassette transporter Yeast 20 575-585

17 Balzi E Wang M Leterme S Van Dyck L and Goffeau A (1994) PDR5 a

novel yeast multidrug resistance conferring transporter controlled by the

transcription regulator PDR1 J Biol Chem 269 2206-2214

85

18 Golin J Ambudkar S V and May L (2007) The yeast Pdr5p multidrug

transporter how does it recognize so many substrates Biochem Biophys Res

Commun 356 1-5

19 Rogers B Decottignies A Kolaczkowski M Carvajal E Balzi E and

Goffeau A (2001) The pleitropic drug ABC transporters from Saccharomyces

cerevisiae J Mol Microbiol Biotechnol 3 207-214

20 Bartosiewicz D and Krasowska A (2009) Inhibitors of ABC transporters and

biophysical methods to study their activity Z Naturforsch C 64 454-458

21 Akache B MacPherson S Sylvain M A and Turcotte B (2004) Complex

interplay among regulators of drug resistance genes in Saccharomyces cerevisiae

J Biol Chem 279 27855-27860

22 Ferreira-Pereira A Marco S Decottignies A Nader J Goffeau A and

Rigaud J L (2003) Three-dimensional reconstruction of the Saccharomyces

cerevisiae multidrug resistance protein Pdr5p J Biol Chem 278 11995-11999

23 Ernst R Kueppers P Stindt J Kuchler K and Schmitt L (2009) Multidrug

efflux pumps Substrate selection in ATP-binding cassette multidrug efflux

pumps - first come first served FEBS J

24 Piper P Mahe Y Thompson S Pandjaitan R Holyoak C Egner R

Muhlbauer M Coote P and Kuchler K (1998) The pdr12 ABC transporter is

required for the development of weak organic acid resistance in yeast EMBO J

17 4257-4265

25 Chloupkova M Reaves S K LeBard L M and Koeller D M (2004) The

mitochondrial ABC transporter Atm1p functions as a homodimer FEBS Lett 569

65-69

26 Kispal G Csere P Prohl C and Lill R (1999) The mitochondrial proteins

Atm1p and Nfs1p are essential for biogenesis of cytosolic FeS proteins EMBO J

18 3981-3989

27 Young L Leonhard K Tatsuta T Trowsdale J and Langer T (2001) Role

of the ABC transporter Mdl1 in peptide export from mitochondria Science 291

2135-2138

28 Chloupkova M LeBard L S and Koeller D M (2003) MDL1 is a high copy

suppressor of ATM1 evidence for a role in resistance to oxidative stress J Mol

Biol 331 155-165

29 Dean M Allikmets R Gerrard B Stewart C Kistler A Shafer B

Michaelis S and Strathern J (1994) Mapping and sequencing of two yeast

genes belonging to the ATP-binding cassette superfamily Yeast 10 377-383

30 Haimeur A Conseil G Deeley R G and Cole S P (2004) The MRP-related

and BCRPABCG2 multidrug resistance proteins biology substrate specificity

and regulation Curr Drug Metab 5 21-53

31 Mason D L and Michaelis S (2002) Requirement of the N-terminal extension

for vacuolar trafficking and transport activity of yeast Ycf1p an ATP-binding

cassette transporter Mol Biol Cell 13 4443-4455

32 Paumi C M Menendez J Arnoldo A Engels K Iyer K R Thaminy S

Georgiev O Barral Y Michaelis S and Stagljar I (2007) Mapping protein-

protein interactions for the yeast ABC transporter Ycf1p by integrated split-

ubiquitin membrane yeast two-hybrid analysis Mol Cell 26 15-25

86

33 Cui Z Hirata D Tsuchiya E Osada H and Miyakawa T (1996) The

multidrug resistance-associated protein (MRP) subfamily (Yrs1Yor1) of

Saccharomyces cerevisiae is important for the tolerance to a broad range of

organic anions J Biol Chem 271 14712-14716

34 Schuller C Mamnun Y M Mollapour M Krapf G Schuster M Bauer B

E Piper P W and Kuchler K (2004) Global phenotypic analysis and

transcriptional profiling defines the weak acid stress response regulon in

Saccharomyces cerevisiae Mol Biol Cell 15 706-720

35 Holyoak C D Bracey D Piper P W Kuchler K and Coote P J (1999) The

Saccharomyces cerevisiae weak-acid-inducible ABC transporter Pdr12 transports

fluorescein and preservative anions from the cytosol by an energy-dependent

mechanism J Bacteriol 181 4644-4652

36 Gregori C Bauer B Schwartz C Kren A Schuller C and Kuchler K

(2007) A genetic screen identifies mutations in the yeast WAR1 gene linking

transcription factor phosphorylation to weak-acid stress adaptation FEBS J 274

3094-3107

37 Papadimitriou M N Resende C Kuchler K and Brul S (2007) High Pdr12

levels in spoilage yeast (Saccharomyces cerevisiae) correlate directly with sorbic

acid levels in the culture medium but are not sufficient to provide cells with

acquired resistance to the food preservative Int J Food Microbiol 113 173-179

38 Tarassov K Messier V Landry C R Radinovic S Serna Molina M M

Shames I Malitskaya Y Vogel J Bussey H and Michnick S W (2008) An

in vivo map of the yeast protein interactome Science 320 1465-1470

39 Ketchum C J Schmidt W K Rajendrakumar G V Michaelis S and

Maloney P C (2001) The yeast a-factor transporter Ste6p a member of the ABC

superfamily couples ATP hydrolysis to pheromone export J Biol Chem 276

29007-29011

40 McGrath J P and Varshavsky A (1989) The yeast STE6 gene encodes a

homologue of the mammalian multidrug resistance P-glycoprotein Nature 340

400-404

41 Kelm K B Huyer G Huang J C and Michaelis S (2004) The internalization

of yeast Ste6p follows an ordered series of events involving phosphorylation

ubiquitination recognition and endocytosis Traffic 5 165-180

42 Loayza D Tam A Schmidt W K and Michaelis S (1998) Ste6p mutants

defective in exit from the endoplasmic reticulum (ER) reveal aspects of an ER

quality control pathway in Saccharomyces cerevisiae Mol Biol Cell 9 2767-

2784

43 Loayza D and Michaelis S (1998) Role for the ubiquitin-proteasome system in

the vacuolar degradation of Ste6p the a-factor transporter in Saccharomyces

cerevisiae Mol Cell Biol 18 779-789

44 Kuchler K Sterne R E and Thorner J (1989) Saccharomyces cerevisiae STE6

gene product a novel pathway for protein export in eukaryotic cells EMBO J 8

3973-3984

45 Teem J L Berger H A Ostedgaard L S Rich D P Tsui L C and Welsh

M J (1993) Identification of revertants for the cystic fibrosis delta F508 mutation

using STE6-CFTR chimeras in yeast Cell 73 335-346

87

46 Elion E A (2000) Pheromone response mating and cell biology Curr Opin

Microbiol 3 573-581

47 Yu R C Pesce C G Colman-Lerner A Lok L Pincus D Serra E Holl

M Benjamin K Gordon A and Brent R (2008) Negative feedback that

improves information transmission in yeast signalling Nature 456 755

48 Metzger M B and Michaelis S (2009) Analysis of quality control substrates in

distinct cellular compartments reveals a unique role for Rpn4p in tolerating

misfolded membrane proteins Mol Biol Cell 20 1006-1019

49 Fromont-Racine M Mayes A E Brunet-Simon A Rain J C Colley A

Dix I Decourty L Joly N Ricard F Beggs J D and Legrain P (2000)

Genome-wide protein interaction screens reveal functional networks involving

Sm-like proteins Yeast 17 95-110

50 Miller J P Lo R S Ben-Hur A Desmarais C Stagljar I Noble W S and

Fields S (2005) Large-scale identification of yeast integral membrane protein

interactions Proc Natl Acad Sci U S A 102 12123-12128

51 Adle D J Wei W Smith N Bies J J and Lee J (2009) Cadmium-mediated

rescue from ER-associated degradation induces expression of its exporter Proc

Natl Acad Sci U S A 106 10189-10194

52 Berkower C Taglicht D and Michaelis S (1996) Functional and physical

interactions between partial molecules of STE6 a yeast ATP-binding cassette

protein J Biol Chem 271 22983-22989

53 Suter B Kittanakom S and Stagljar I (2008) Two-hybrid technologies in

proteomics research Curr Opin Biotechnol 19 316-323

54 Lalonde S Ehrhardt D W Loque D Chen J Rhee S Y and Frommer W

B (2008) Molecular and cellular approaches for the detection of protein-protein

interactions latest techniques and current limitations The Plant Journal 53 610-

635

55 Fields S and Song O (1989) A novel genetic system to detect protein-protein

interactions Nature 340 245-246

56 Fields S (2009) Interactive learning lessons from two hybrids over two decades

Proteomics 9 5209-5213

57 Koegl M and Uetz P (2007) Improving yeast two-hybrid screening systems

Brief Funct Genomic Proteomic 6 302-312

58 Auerbach D Thaminy S Hottiger M O and Stagljar I (2002) The post-

genomic era of interactive proteomics facts and perspectives Proteomics 2 611-

623

59 Ito T Chiba T Ozawa R Yoshida M Hattori M and Sakaki Y (2001) A

comprehensive two-hybrid analysis to explore the yeast protein interactome Proc


60 Uetz P Giot L Cagney G Mansfield T A Judson R S Knight J R

Lockshon D Narayan V Srinivasan M Pochart P Qureshi-Emili A Li Y

Godwin B Conover D Kalbfleisch T Vijayadamodar G Yang M

Johnston M Fields S and Rothberg J M (2000) A comprehensive analysis of

protein-protein interactions in Saccharomyces cerevisiae Nature 403 623-627

61 Lievens S Lemmens I and Tavernier J (2009) Mammalian two-hybrids come

of age Trends Biochem Sci 34 579-588

88

62 Stagljar I Korostensky C Johnsson N and te Heesen S (1998) A genetic

system based on split-ubiquitin for the analysis of interactions between membrane

proteins in vivo Proc Natl Acad Sci U S A 95 5187-5192

63 Glickman M H and Ciechanover A (2002) The ubiquitin-proteasome

proteolytic pathway destruction for the sake of construction Physiol Rev 82 373-

428

64 Hershko A and Ciechanover A (1992) The ubiquitin system for protein

degradation Annu Rev Biochem 61 761-807

65 Verma R Oania R Graumann J and Deshaies R J (2004) Multiubiquitin

chain receptors define a layer of substrate selectivity in the ubiquitin-proteasome

system Cell 118 99-110

66 Johnsson N and Varshavsky A (1994) Split ubiquitin as a sensor of protein

interactions in vivo Proc Natl Acad Sci U S A 91 10340-10344

67 Iyer K Burkle L Auerbach D Thaminy S Dinkel M Engels K and

Stagljar I (2005) Utilizing the split-ubiquitin membrane yeast two-hybrid system

to identify protein-protein interactions of integral membrane proteins Sci STKE

2005 pl3

68 Paumi C M Chuk M Chevelev I Stagljar I and Michaelis S (2008)

Negative regulation of the yeast ABC transporter Ycf1p by phosphorylation

within its N-terminal extension J Biol Chem 283 27079-27088

69 Thaminy S Auerbach D Arnoldo A and Stagljar I (2003) Identification of

novel ErbB3-interacting factors using the split-ubiquitin membrane yeast two-

hybrid system Genome Res 13 1744-1753

70 Fetchko M and Stagljar I (2004) Application of the split-ubiquitin membrane

yeast two-hybrid system to investigate membrane protein interactions Methods

32 349-362

71 Condamine T Le Texier L Howie D Lavault A Hill M Halary F

Cobbold S Waldmann H Cuturi M C and Chiffoleau E (2010) Tmem176B

and Tmem176A are associated with the immature state of dendritic cells J

Leukoc Biol 1-9

72 Regan J A and Laimins L A (2008) Bap31 is a novel target of the human

papillomavirus E5 protein J Virol 82 10042-10051

73 Ferrandiz-Huertas C Fernandez-Carvajal A and Ferrer-Montiel A (2010)

RAB4 interacts with the human P-glycoprotein and modulates its surface

expression in multidrug resistant K562 cells Int J Cancer

74 Gisler S M Kittanakom S Fuster D Wong V Bertic M Radanovic T

Hall R A Murer H Biber J Markovich D Moe O W and Stagljar I

(2008) Monitoring protein-protein interactions between the mammalian integral

membrane transporters and PDZ-interacting partners using a modified split-

ubiquitin membrane yeast two-hybrid system Mol Cell Proteomics 7 1362-1377

75 Scheper W Thaminy S Kais S Stagljar I and Romisch K (2003)

Coordination of N-glycosylation and protein translocation across the endoplasmic

reticulum membrane by Sss1 protein J Biol Chem 278 37998-38003

76 Deribe Y L Wild P Chandrashaker A Curak J Schmidt M H

Kalaidzidis Y Milutinovic N Kratchmarova I Buerkle L Fetchko M J

Schmidt P Kittanakom S Brown K R Jurisica I Blagoev B Zerial M

89

Stagljar I and Dikic I (2009) Regulation of epidermal growth factor receptor

trafficking by lysine deacetylase HDAC6 Sci Signal 2 ra84

77 Kittanakom S Chuk M Wong V Snider J Edmonds D Lydakis A

Zhang Z Auerbach D and Stagljar I (2009) Analysis of Membrane Protein

Complexes Using the Split-Ubiquitin Membrane Yeast Two-Hybrid (MYTH)

System in Yeast Functional Genomics and Proteomics Methods and Protocols

(Stagljar I Ed) p 247 Humana Press New York

78 Inoue H Nojima H and Okayama H (1990) High efficiency transformation of

Escherichia coli with plasmids Gene 96 23-28

79 Winzeler E A Shoemaker D D Astromoff A Liang H Anderson K

Andre B Bangham R Benito R Boeke J D Bussey H Chu A M

Connelly C Davis K Dietrich F Dow S W El Bakkoury M Foury F

Friend S H Gentalen E Giaever G Hegemann J H Jones T Laub M

Liao H Liebundguth N Lockhart D J Lucau-Danila A Lussier M

MRabet N Menard P Mittmann M Pai C Rebischung C Revuelta J L

Riles L Roberts C J Ross-MacDonald P Scherens B Snyder M Sookhai-

Mahadeo S Storms R K Veronneau S Voet M Volckaert G Ward T R

Wysocki R Yen G S Yu K Zimmermann K Philippsen P Johnston M

and Davis R W (1999) Functional characterization of the S cerevisiae genome

by gene deletion and parallel analysis Science 285 901-906

80 Chen D C Yang B C and Kuo T T (1992) One-step transformation of yeast

in stationary phase Curr Genet 21 83-84

81 Shimomura T Ando S Matsumoto K and Sugimoto K (1998) Functional

and physical interaction between Rad24 and Rfc5 in the yeast checkpoint

pathways Mol Cell Biol 18 5485-5491

82 Rockwell N C Wolfger H Kuchler K and Thorner J (2009) ABC

transporter Pdr10 regulates the membrane microenvironment of Pdr12 in

Saccharomyces cerevisiae J Membr Biol 229 27-52

83 Hama H Tall G G and Horazdovsky B F (1999) Vps9p is a guanine

nucleotide exchange factor involved in vesicle-mediated vacuolar protein

transport J Biol Chem 274 15284-15291

84 Hillenmeyer M E Fung E Wildenhain J Pierce S E Hoon S Lee W

Proctor M St Onge R P Tyers M Koller D Altman R B Davis R W

Nislow C and Giaever G (2008) The chemical genomic portrait of yeast

uncovering a phenotype for all genes Science 320 362-365

85 Balzi E and Goffeau A (1995) Yeast multidrug resistance the PDR network J

Bioenerg Biomembr 27 71-76

86 Gregori C Schuller C Frohner I E Ammerer G and Kuchler K (2008)

Weak organic acids trigger conformational changes of the yeast transcription

factor War1 in vivo to elicit stress adaptation J Biol Chem 283 25752-25764

87 Wolfger H Mahe Y Parle-McDermott A Delahodde A and Kuchler K

(1997) The yeast ATP binding cassette (ABC) protein genes PDR10 and PDR15

are novel targets for the Pdr1 and Pdr3 transcriptional regulators FEBS Lett 418

269-274

88 Wilcox L J Balderes D A Wharton B Tinkelenberg A H Rao G and

Sturley S L (2002) Transcriptional profiling identifies two members of the ATP-

90

binding cassette transporter superfamily required for sterol uptake in yeast J Biol

Chem 277 32466-32472

89 Burd C G Mustol P A Schu P V and Emr S D (1996) A yeast protein

related to a mammalian Ras-binding protein Vps9p is required for localization of

vacuolar proteins Mol Cell Biol 16 2369-2377

90 Kolaczkowska A Kolaczkowski M Goffeau A and Moye-Rowley W S

(2008) Compensatory activation of the multidrug transporters Pdr5p Snq2p and

Yor1p by Pdr1p in Saccharomyces cerevisiae FEBS Lett 582 977-983

91 Raths S Rohrer J Crausaz F and Riezman H (1993) end3 and end4 two

mutants defective in receptor-mediated and fluid-phase endocytosis in

Saccharomyces cerevisiae J Cell Biol 120 55-65

92 Vojtek A B Hollenberg S M and Cooper J A (1993) Mammalian Ras

interacts directly with the serinethreonine kinase Raf Cell 74 205-214

93 Kelly D E Lamb D C and Kelly S L (2001) Genome-wide generation of

yeast gene deletion strains Comp Funct Genomics 2 236-242

94 Brachmann C B Davies A Cost G J Caputo E Li J Hieter P and

Boeke J D (1998) Designer deletion strains derived from Saccharomyces

cerevisiae S288C a useful set of strains and plasmids for PCR-mediated gene

disruption and other applications Yeast 14 115-132

91

APPENDIX

92

Appendix I ndash Yeast Strains Media Recipes and Reagents


Strain Genotype Source

L40 MATa trp1 leu2 his3 LYS2lexA-HIS3 URA3lexALacZ (92)

PDR12-CYT MATa PDR12-CYT (isogenic to L40) I Stagljar (University of

Toronto Toronto)

PDR12-CT MATa PDR12-CT (isogenic to L40) I Stagljar

STE6-CYT MATa STE6-CYT (isogenic to L40) I Stagljar

STE6-CT MATa STE6-CT (isogenic to L40) I Stagljar

BY157 MATa gcn2-101 ura3-52 C Nislow (University of

Toronto Toronto)

BY158 MATα gcn2-101 gcn3-101 ura3-52 C Nislow

BY4741 MATa ura3D leu2D his3D met15 D LYS2 (93)

BY4742 MATa ura3D leu2 his3D MET15 lys2D (93)

BY4743 MATaα his3Δ1his3Δ1 leu2Δ0leu2Δ0 LYS2lys2Δ0

met15Δ0MET15 ura3Δ0ura3Δ0

(94)

DDK1240 MATa pdr12Δkan (isogenic to L40) This study

DDN1240 MATa pdr12Δnat (isogenic to L40) This study

DDK0640 MATa ste6Δkan (isogenic to L40) This study

DDS0640 MATa sac6Δnat STE6-CYT (isogenic to L40)

DDN1242 MATa pdr12Δnat (isogenic to BY4742) This study

DD1210 MATaα pdr10Δkan pdr12Δnat (isogenic to BY4743) This study



DDG121 MATaα gtt1Δkan pdr12Δnat (isogenic to BY4743) This study

DDS121 MATaα sod1Δkan pdr12Δnat (isogenic to BY4743) This study

DD1207 MATaα tma7Δkan pdr12Δnat (isogenic to BY4743) This study

DD1256 MATaα ybr056wΔkan pdr12Δnat (isogenic to BY4743) This study

DDZ121 MATaα zeo1Δkan pdr12Δnat (isogenic to BY4743) This study


Plasmid Features Promoter Resistance Marker Source

L2 Cub-TF-KanMX AMPR

DSB

L3 TF-Cub-KanMX AMPR DSB

pPR3N NubG-HA ADH TRP1 AMPR DSB

p4339 Nat Cassette T7 AMPR NAT

R

pFur4-NubG Fur4-HA-NubG ADH TRP1 AMPR DSB

pFur4-NubI Fur4-HA-NubI ADH TRP1 AMPR DSB

pOst1-NubG Ost1-HA-NubG ADH TRP1 AMPR DSB

93

pOst1-NubI Ost1-HA-NubI ADH TRP1 AMPR DSB

pDONR223 attB1 and attB2 T7 SPCR

Invitrogen

pYES-DEST52 V5 ndash HIS6 Epitope GAL1 T7 URA3 AMPR Invitrogen

DBS ndash Dual Systems Biotech

Recipes

05M EDTA pH 80

Dissolve 9305 g of EDTA (disodium salt dihydrate) in 400 mL of ddH2O Adjust pH to

80 using NaOH pellets and bring the final volume up to 500 mL with ddH2O Autoclave

and store at room temperature

09 NaCl

Dissolve 09 g of NaCl in a final volume of 100 mL of ddH2O Autoclave and store at

room temperature

1M 3-AT Solution

Dissolve 84 g of 3-Amino-124-triazole (3-AT) in a total volume of 100 mL ddH2O

Filter sterilize and aliquot as required Store at -20degC

1M Lithium Acetate

Dissolve 102 g of lithium acetate dihydrate in a total volume of 100 mL of ddH2O

Autoclave and store at room temperature

1M Tris pH 75

Dissolve 12114 g of Tris Base in 800 mL ddH2O Adjust the pH to 75 using

concentrated HCl and bring the volume up to 1L with ddH2O Autoclave and store at

room temperature

10X Tris EDTA (TE) Buffer pH 75

Mix 100 mL of 1M Tris pH 75 20 mL of 05M EDTA pH 80 and 880 mL of ddH2O

Filter sterilize through a 02 microm pore filter and store at room temperature

10x Drop-out Mix

Dissolve the appropriate amino acids in a total volume of 2 L of ddH2O Autoclave and

store at 4degC Omit components from the above solution as required depending upon the

selective medium being prepared

94

Amino Acid 2L (mg)

Isoleucine 600

Valine 3000

Adenine (A) 800

Histidine (H) 400

Leucine 2000

Lysine 600

Methionine 3000

Phenylalanine 1000

Threonine 4000

Tryptophan (W) 800

Tyrosine 600

Uracil 400

Arginine 400

50 PEG Solution (wv)

Dissolve 50 g of PEG-3350 in a total volume of 100 mL of ddH2O Autoclave or filter

sterilize once completely dissolved Make fresh

Ampicillin (1000x) Stock

Dissolve 100 mg of Ampicillin sodium salt in a total volume of 1 mL of ddH2O Filter

sterilize and aliquot as required Use at a working concentration of 100 mgL Store at -

20degC

Geneticin (G418) (1000x) Stock

Dissolve 200 mg of G418 sulphate in a total volume of 1 mL ddH2O Filter sterilize and

aliquot as required Use at a working concentration of 200 mgL Store at 4degC

Kanamycin (1000x) Stock

Dissolve 50 mg of Kanamycin monosulphate in a total volume of 1 mL of ddH2O Filter


20degC

Spectinomycin (1000x) Stock

Dissolve 100 mg of Spectinomycin dihydrochloride pentahydrate in a total volume of 1

mL of ddH2O Filter sterilize and aliquot as required Use at a working concentration of

100 mgL Store at -20degC

Transformation Master Mix

Per reaction combine 240 μL sterile 50 PEG 36 μL 1M LiOAc and 25 μL ssDNA

Vortex well to combine and use immediately Do not store for later use

Single-stranded Carrier DNA (ssDNA) Solution

Sterilize a 250 mL bottle and magnetic stir bar by autoclaving Dissolve 200 mg of

salmon sperm DNA in 100 mL sterile ddH2O Aliquot solution into sterile 15 mL

95

microfuge tubes Boil at 100degC for 5 minutes and put on ice immediately Store at -

20degC Before use boil again for 5 min at 100degC

Sodium Phosphate Solution

Dissolve 7 g of sodium phosphate dibasic and 3 g of sodium phosphate monobasic in a

total volume of 100 mL of ddH2O Autoclave and store at room temperature

X-Gal Solution

Dissolve 100 mg of X-Gal powder in a 1 mL total volume of NN-dimethyl formamide

Make fresh just before use Do not expose to light for prolonged periods of time

LB +- Antibiotic Medium (Liquid and Solid)

Dissolve 10 g bio-tryptone 5 g yeast extract and 10 g of NaCl in a total volume of 1 L of

ddH2O If making solid medium add 15 g Agar Autoclave and store liquid medium at

room temperature adding antibiotic (if required) before use at the appropriate working

concentration For solid medium allow to cool to 50degC add antibiotic (if required) at the

appropriate working concentration and pour into sterile petri dishes Store at 4degC

Synthetic Dropout (SD) Medium (Liquid and Solid)

Dissolve 67 g of yeast nitrogen base 20 g of D-glucose and 20 g of agar (omit if

preparing liquid medium) in a total volume of 900 mL of ddH2O Add 100 mL of the

appropriate 10X Drop-out Mix Autoclave and store liquid medium at room temperature

For solid medium allow to cool to 50degC and pour into sterile petri dishes Store at 4degC

If inclusion of 3-AT in the solid medium is required reduce the initial volume of ddH2O

by the volume of 1M 3-AT solution needed to obtain the desired concentration Add 3-

AT solution after autoclaving once the medium has cooled to 50C

Synthetic Dropout (SD) + X-Gal Medium (Solid)

Dissolve 67 g of yeast nitrogen base 20 g of D-glucose and 20 g of agar in a total

volume of 800 mL of ddH2O Add 100 mL of the appropriate 10X Drop-out Mix

Autoclave allow to cool to 50degC then add 100 mL of sodium phosphate solution and 800

microL of X-Gal solution Mix and pour into sterile petri dishes Wrap in aluminum foil and

store at 4degC If inclusion of 3-AT in the solid medium is required reduce the initial

volume of ddH2O by the volume of 1M 3-AT solution needed to obtain the desired

concentration Add 3-AT solution after autoclaving once the medium has cooled to

50C X-Gal is light sensitive therefore do not expose plates to light for prolonged

periods of time

YPAD +- Antibiotic Medium (Liquid and Solid)

Dissolve 10 g of yeast extract 20 g peptone 20 g of D-glucose 40 mg of adenine

sulphate and 20 g of agar (omit if preparing liquid medium) in a total volume of 1 L of

ddH2O Autoclave and store liquid medium at room temperature adding antibiotic (if

required) at the appropriate working concentration before use Cool solid medium to

50degC before adding antibiotic (if required) at the appropriate working concentration and

pour into sterile petri dishes Store at 4degC

96

2X YPAD (Liquid Medium)

Dissolve 20 g of yeast extract 40 g peptone 40 g of D-glucose and 40 mg of adenine

sulphate in a total volume of 1 L of ddH2O Autoclave and store at room temperature

Agarose Gel

Mix 1 g agarose in 100 mL 1x TAE Microwave for until solution is clear about 1 and a

half minutes and allow to cool slightly before adding 4 μL of SYBR Safe DNA gel stain

(Invitrogen) Pour into tray and allow to solidify for at least 15 minutes prior to use

1M Sorbitol

Dissolve 455 g D-sorbitol in a total volume of 250 mL of ddH2O Filter sterilize and

store at room temperature

Solution A

Combine 250 mL of 4M sorbitol 100 mL of 1M sodium citrate 120 mL of 05M EDTA

and 530 mL of ddH2O for a tola volume of 1L in a bottle with a magnetic stir bar


Zymolyase Solution (5 mgml in 1M sorbitol)

Combine 0025 g Zymolyase 100T powder and 5 mL 1M sorbitol Store at 4˚C until

needed

Lysis Solution

Combine 20 mL of Solution A 45 mL of Zymolyase solution and 220 μL β-

mercaptoethanol Use immediately after preparation

Terrific Broth (TB)

Dissolve 12 g of tryptone 24 g of yeast extract and 4 mL 100 glycerol in 900 mL of

ddH2O Autoclave then add 100 mL sterile solution of 017M KH2PO4 and 072M

K2HPO4 which is made by dissolving 231 g of KH2PO4 and 1254 g of K2HPO4 in a

total volume of 100 mL of ddH2O Before use add antibiotic (if required) at the

appropriate working concentration

T-B Buffer

Dissolve 1088 g of MnCl24H2O 220 g of CaCl22H2O and 1865 g of KCl in 900 mL

of ddH2O Add 20 mL PIPES (05M pH 67) and top up to 1 L with ddH2O Filter

sterilize and store at -20˚C in 50 mL aliquots until required

Sporulation Medium

Dissolve 10 g of potassium acetate (1) 1 g of yeast extract (01) 05 g of glucose

(005) and 20 g of agar (2) in up to 1 L of ddH2O Autoclave cool to about 55˚C and

pour plates Store at 4˚C

97

SD Minimal Plates


volume of 1 L of ddH2O Autoclave and allow to cool to 50degC then pour into sterile

petri dishes Store at 4degC

Sorbic Acid Solid Medium


sulphate 10 g of agar and 56 mg of Sorbic acid per mM in a total volume of 500 mL of

ddH2O Autoclave and cool the medium to 50degC before pouring into sterile petri dishes

Store at 4degC

Benzoic Acid Solid Medium


sulphate 10 g of agar and 61 mg of Benzoic acid per mM in a total volume of 500 mL of


Store at 4degC

1M Stock of Sorbic Acid

Dissolve 56 g of Sorbic acid in a total volume of 50 mL of 100 ethanol Vortex

vigorously until solution is completely clear Store at room temperature

1M Stock of Benzoic Acid

Dissolve 61 g of Benzoic acid in a total volume of 50 mL of 100 ethanol Vortex


YPAD +Acid Liquid Medium

To make stock solutions of YPAD containing various concentrations of either Sorbic or

Benzoic acid add the amount of 1M stock acid solution indicated in the table below to a

total volume of 50 mL YPAD Vortex to combine and store at room temperature

1M Acid Stock Added Stock YPAD + Acid

Medium

Working Concentration Total Volume

1000 microL 20 mM 10 mM 50 mL






Please note that for the liquid panelling assay 50 microL of cells are added to each well halving the stock

solution of YPAD + Acid into the desired working concentration

4X Separating Buffer pH 87

Combine 6055 g of Tris base (15M) and 2 g of SDS (04) in a final volume of 500 mL

of ddH2O Adjust the pH to 87 by adding concentrated HCl Store at room temperature

98

4X Stacking Buffer pH 68



8 Acrylamide SDS PAGE Gels

For the separating gel combine 937 mL of 4X separating buffer 181 mL of ddH2O 10

mL of 30 acrylamide 50 microL of TMED and 250 microL of 10 APS solution Pour into

casts and add 400 microL of isopropanol along the top Once set prepare the stacking gel

mix by adding 25 mL of 4X stacking buffer 61 mL of ddH2O 134 mL of 30

acrylamide 20 microL of TMED and 100 microL of 10 APS Pour into casts add combs and

allow to set If storing gels for later use wrap in wet paper towels and place in a plastic

bag at 4degC Makes four gels

10 APS Solution

Dissolve 1 g of APS in 10 mL of ddH2O Store at 4degC

10X TBS pH 75

Dissolve 6055 g of Tris base (50 mM) and 8766 g of NaCl (150 mM) in a final volume

of 1 L of ddH2O Adjust the pH to 75 by adding concentrated HCl and store at 4degC

1X TBST Solution

Mix 100 mL of 10X TBS solution with 900 mL of ddH2O Add 1 mL of Tween 20 and

mix well Store at room temperature

Blocking and Incubation Solutions

Dissolve 5 g of skim milk powder in 100 mL of 1X TBST solution to make 5 milk

TBST for blocking Dissolve 02 g of skim milk powder in 20 mL of 1X TBST to make

1 milk TBST solution for the primary antibody incubation Dissolve 002 g of skim

milk powder in 20 mL of 1X TBST to make 01 milk TBST solution for secondary

antibody incubation

Antibodies

Polyclonal rabbit α-VP16 1deg antibody

Monoclonal mouse α-LexA 1deg antibody

Polyclonal mouse α-HA 1deg antibody

Monoclonal mouse α-HA 1deg antibody

Monoclonal rat α-HA 1deg antibody

Monoclonal mouse α-V5 1deg antibody

Monoclonal mouse α-HIS 1deg antibody

Sheep anti-mouse horseradish peroxidase (HRP) ndash conjugated

Goat anti-rabbit horseradish peroxidase (HRP) ndash conjugated

Goat anti-rat horseradish peroxidise (HRP) ndash conjugated

99

Appendix II ndash PCR Protocols and Primer Sequences


Bait Generation and Confirmation Primers

ORF Forward Reverse

PDR12 (Int) 5rsquoATTTTCCAAACAGTTCCAGGTGACGAAAATAAA ATCACGAAGAAAATGTCGGGGGGGATCCCTCC 3rsquo

5rsquoACTCACGAGTGGGATAGAAATGAAATTCTTTT CTTTTAAATGGTAACTATAGGGAGACCGGCAG 3rsquo

PDR12 (Conf) 5rsquoGGATCACAGATGGAGAAACTT 3rsquo NA

STE6 (Int) 5rsquoAATAATCGCGGGGAATTATTCCAAATTGTTTCCA

ACCAAAGCAGTATGTCGGGGGGGATCCCTCCA 3rsquo

5rsquoGTCTCGAATATTTGAGTATGTTTTAGTTTTTTG

TTTTATATTTTCACTATAGGGAGACCGGCAGA 3rsquo

STE6 (Conf) 5rsquoTCAGCCTTGGATTCTGTCAG 3rsquo NA

Deletion Confirmation Primers

ORF Forward Reverse

ATG27 5rsquoGGTTAGTGGCATATTAGTCTGCTGT 3rsquo 5rsquoTCTTGCGGTAAATCGTTTATCTTAC 3rsquo

COS8 5rsquoGGCACACCGTGATGCACCCG 3rsquo 5rsquoCATGTTAATGACACCATGGCAG 3rsquo

CYB5 5rsquoAGTGAGAGAGGTTAGCATAACGAGA 3rsquo 5rsquoGATCGTATTGAAGTAAGAGCAGAGC 3rsquo

GTT1 5rsquoCAAATGAGGATTTTTACAAGGCTTA 3rsquo 5rsquoGTTTACAAGTTTTTGAAGAGCCAAA 3rsquo

GUP2 5rsquoCTACTCGTTTACCTGTAATCTTGGC 3rsquo 5rsquoGTCGCAACTTAGTGATGCATATAGA 3rsquo

IKS1 5rsquo TTTTCAGGATCACATAAATGCATAA 3rsquo 5rsquoGCACATTAAGGTATTGTTCGCTATT 3rsquo

LRE1 5rsquoGCTGTAGTGTGTCCTCCAATACTCT 3rsquo 5rsquoCTCCAAGATTACTGAAAAACCTGAA 3rsquo

Nat Int Conf 5rsquoCTTCGTGGTCATCTCGTACTC 3rsquo 5rsquoGAGTACGAGATGACCACGAAG 3rsquo

NCE102 5rsquoTCTTCCTACTTCTTCTTCCATTTCC 3rsquo 5rsquoAATTATAATAAAAGAAAGCGGGGTG 3rsquo

PDR10 5rsquoGTACTACTACAGAATTGGTCGGCAT 3rsquo 5rsquoTCACTGCAGATGTTAATAGATCCAA 3rsquo

PDR11 5rsquoCACTTTTGTTTCCTACAACTTCCAC 3rsquo 5rsquoGATGCAAATCAAGGAATGTTCTAAT 3rsquo

PDR5 5rsquoTTGAACGTAATCTGAGCAATACAAA 3rsquo 5rsquoTCACACTAAATGCTGATGCCTATAA 3rsquo

PHO88 5rsquoAGAAGAAGAACATCACTTTACACGG 3rsquo 5rsquoGGACACGACTCATTTTTCTTTACAT 3rsquo

RHO5 5rsquo TTTCAGTTTCTCGTAGCTTTTCCTA 3rsquo 5rsquoATTTGCTCGTAAAGAATTTGATGAC 3rsquo

SAC6 5rsquoCCGGATATAGGGTCCTATTTTCTTA 3rsquo 5rsquoCATTTTCTGCATATTTCAAAGAACC 3rsquo

SMF2 5rsquoTAGAATGAACCACAAGTTTGTAGCA 3rsquo 5rsquoTAAGTGTGCTAAAATGTGGATGAAA 3rsquo

SOD1 5rsquoGACGTAAGTATCTCTGAAGTGCAGC 3rsquo 5rsquoGGAAGCTTTATGGTGAAGTTAATGA 3

SPC2 5rsquoTGACAATTGTACACGTTGAAACGGAAT 3rsquo 5rsquoTTTGAGGATGCATGATTATAGCCTAGC 3rsquo

STE6 5rsquoACACGCTGCTTCGCACATATAC 3rsquo 5rsquoCCTGCCATCGCAACAACCAC 3rsquo

TAT1 5rsquoAAACTTCACATTATCTTGACAAGGC 3rsquo 5rsquoTTTTCTTGGCACATTTACACACTTA 3rsquo

100

TMA7 5rsquoGGATACAAGATCACCCATCATAAAG 3rsquo 5rsquoATATTTATCCTTATGCCTGTCACCA 3rsquo

YBR056W 5rsquoAGCTACTAAAGAAAGAGTGCTGCAA 3rsquo 5rsquoCTTCATCTTGATTACCATTATTCCG 3rsquo

YCK2 5rsquoTGTCTCCACAAAATGAGTAATGAAA 3rsquo 5rsquoATAATATTGGCGCTTCCTTAAGAGT 3rsquo

YGL082W 5rsquoTATCTTAAATTGGCTTGAAACGAAC 3rsquo 5rsquoTTCTGTGAAGATATCCCAAAAATGT 3rsquo

YLL023C 5rsquoTGACTTCAATGATCTCTCTCAACTG 3rsquo 5rsquoAAAAAGCTTCGGAAATACTACGAAT 3rsquo

YLR154C-G 5rsquoTAGACCGTAAGGTCGGGTCG 3rsquo 5rsquoCACGCAAGTAGTCCGCCTAG 3rsquo

YML133C 5rsquoCAGGCCGGAAATCAAGGATG 3rsquo 5rsquoGTACGTCTCCTCCAAGCCCT 3rsquo

YOP1 5rsquo GTAAGTAGGTTATATGGCTGCTGGA 3rsquo 5rsquoATAACATGATTAATGACCTTGCGTT 3rsquo

YSY6 5rsquoAATAATGGAAGTGAAACAAGGCTAA 3rsquo 5rsquoAAAGCAGAAAGCCTACTTGAAAAAT 3rsquo

ZEO1 5rsquoGCTTTATCGTGTTTTATATCGATGG 3rsquo 5rsquoGATTCTCGTACCGCTCATATTTTTA 3rsquo

ZRT1 5rsquoAAAACAATACACCCGTACTCTCTTG 3rsquo 5rsquoTGAAGCAAACTAGGTCTGTTGTAGA 3rsquo

ZRT3 5rsquoTTGACACATCTCTAAGCTGAAACTG 3rsquo 5rsquoTTGAACATACTCTAAACTCGGGAAC 3rsquo

Deletion Generation Primers

COS8 5rsquoGTTACTGAGCCATTGCATGAACGCGCGCGC

CTCGGCGGCTTGTCGCCCGTACATTTAGCC 3rsquo

5rsquoTCTGCCGGTCTCCCTATAGTCAAATATTGAAAAT

AAGTGTTTTTGAATTTAGTG GTTATTGTATGGTG 3rsquo

PDR12 5rsquoGGTTTACAGATTTATTGTTATTGTTCTTATT AATAAAAAATGTCGCCCGTACATTTAGCC 3rsquo

5rsquoATTGTGTGTTAAACCACGAAATACAAATATA TTTGCTTGCTTGTACTATAGGGAGACCGGCAGA 3rsquo

SAC6 5rsquoGGATATAGGGTCCTATTTTCTTACGTGAACGG

CTTTTCTTCTTGCAGA ATACCCTCCTTGACAGTC 3rsquo

5rsquoGTAGGTGGAAGTTGAAATCTATTATTACATATTA

AAAACTTCGCGACC AGCATTCACATACG 3rsquo

SOD1 5rsquoGTAAGCGGACATCCCTTCCGCTGGGCTCG CCATCGCAGTGTC GCCCGTACATTTAGCC 3rsquo

5rsquoTCTGCCGGTCTCCCTATAGTGACATAAATCTAA GCGAGGGAAATGAAAATG AAT GAATTG 3rsquo

STE6 5rsquoAGTGCCGCTGAAAATTCCACTAGGAAACAAAG

AACAAGCTACGTCTGTCGCCCGTACATTTAGCC 3rsquo

5rsquoTTAACTGCTTTGGTTGGAAACAATTTGGAATAATTC

CCCGCGATTACTATAGGGAGACCGGCAGA 3rsquo

TMA7 5rsquoAATGAACGAGGAAAATAAAAAATTTCATG

TTTAAAATCCTTGTCGCCCGTACAT TTAGCC 3rsquo

5rsquoTCTGCCGGTCTCCCTATAGTAATATATGTA

TTTACTTAAAAAACGAGA ACTAGAAAATAC 3rsquo

YLR154C-G 5rsquoCTCCGTTTCAAAGGCCTGATTTTATGCAGGCCA CCATCGAAAGGGTGTCGCCCGTACATTTAGCC 3rsquo

5rsquoTCTGCCGGTCTCCCTATAGTCTACATTATTCTATC AACTAGAGGCTGT TCACCTTGGAGACCTGC 3rsquo

YML133C 5rsquoCTTCTTCTCAATAGAGTAGCTTAATTATTACA

TTCTTAGATGATGTGT CGCCCGTACATTTAGCC 3rsquo

5rsquoTCTGCCGGTCTCCCTATAGTTGCAACAAACACT

AAATCAAAACAGTGA AATACTACTACATCAAA 3rsquo

Gap Repair Primers

PDR5 NubG 5rsquoTGTTCCAGATTACGCTGGATCCAA

GCAGTGGTATCAACGCAGAGTGATG

CCCGAGGCCAAGCTTAAC 3rsquo

5rsquoCGATAAGCTTGATATCGAATTCTCGA

GAGGCCGAGGCGGCCGACATTATTTCT

TGGAGAGTTTACCG 3rsquo

101

PDR5

5rsquoTCTATAGACACGCAAACACAAATA

CACACACTAATCTAGAACTAGTATGT ACCCATACGATGTTCCAGATTACGCTA

TGCCCGAGGCCAAGCTTAAC 3rsquo

5rsquoCGATAAGCTTGATATCGAATTCTCGAG

AGGCCGAGGCGGCCGACATTATTTCTTG

GAGAGTTTACCG 3rsquo

PDR10 NubG 5rsquoTGTTCCAGATTACGCTGGATCCAAG

CAGTGGTATCAACGCAGAGTGATGTT

GCAAGCGCCCTCAAGTTC 3rsquo


AGGCCGAGGCGGCCGACATTATTTCTTTA

ATTTTTTGCTTTTCTTTG 3rsquo

PDR10


CACACACTAATCTAGAACTAGTATGT

ACCCATACGATGTTCCAGATTACGCTA TGTTGCAAGCGCCCTCAAGTTC 3rsquo

5rsquoCGATAAGCTTGATATCGAATTCTCGAG AGGCCGAGGCGGCCGACATTATTTCTTTA


PDR11 NubG 5rsquoTGTTCCAGATTACGCTGGATCCAAG CAGTGGTATCAACGCAGAGTGATGTC

TCTTTCCAAATATTTTAATCC 3rsquo

5rsquoCGATAAGCTTGATATCGAATTCTCGAG AGGCCGAGGCGGCCGACATTATACGCTT

TGTTCGTTTGG 3rsquo

PDR11

5rsquoTCTATAGACACGCAAACACAAATA CACACACTAATCTAGAACTAGTATGT

ACCCATACGATGTTCCAGATTACGCTA

TGTCTCTTTCCAAATATTTTAATCC 3rsquo


AGGCCGAGGCGGCCGACATTATACGCTT TGTTCGTTTGG 3rsquo

Sequencing Primers

PDR5 NubG 5rsquoAACATGTATGCCCGAGG 3rsquo NA

PDR5 1 5rsquoAGATTACGCTATGCCCGAGG 3rsquo NA

PDR5 2 5rsquoAGGCTCTGGCTGTACTAC 3rsquo NA

PDR5 3 5rsquoTGCCACAGTGGCCATCTATC 3rsquo NA

PDR5 4 5rsquoTGGGTAACTGTAGTATGGC 3rsquo NA

PDR5 5 5rsquoGAATATGTTCCTCGTGGTCC 3rsquo NA

PDR5 6 5rsquoCACTTCTGGATTGTTTGGCC 3rsquo NA

PDR5 7 5rsquoAAGTTGTTGGTGCAGCTC 3rsquo NA

PDR5 8 5rsquoTTTACTCCAACGCGTCTG 3rsquo NA

PDR5 9 5rsquoACTGGTTAGCAAGAGTGCC 3rsquo NA

PDR12 1 5rsquoATGTCTTCGACTGACGAACA 3rsquo NA

PDR12 2 5rsquoTTATTTGTCGTCGGTAGGCC 3rsquo NA

PDR12 3 5rsquoGTTGCTATTTACCAAGCTGG 3rsquo NA

PDR12 4 5rsquoGGGTTAAGGGTGATTCAACG 3rsquo NA

PDR12 5 5rsquoGCATCATTGGATTAGATGGC 3rsquo NA

PDR12 6 5rsquoTACACCATTCCATACGACGG 3rsquo NA

PDR12 7 5rsquoGAGAGCCTTAGCTGATTCTG 3rsquo NA

PDR12 8 5rsquoATCGCCTGTCTATATCAGGG 3rsquo NA

PDR12 9 5rsquoATGCCTGCCTTCTGGAGAAG 3rsquo NA

102

PDR12 10 5rsquoTCCAAACAGTTCCAGGTGAC 3rsquo NA

Gateway Cloning Primers

PDR5 5rsquoGGGGACAAGTTTGTACAAAAAAGC

AGGCTTAATGCCCGAGGCCAAGCTT 3rsquo

5rsquoGGGGACCACTTTGTACAAGAAAGC

TGGGTATTTCTTGGAGAGTTTACC 3rsquo

PDR10 5GGGGACAAGTTTGTACAAAAAAGCA

GGCTTAATGTTGCAAGCGCCCTCAAGT 3rsquo


TGGGTATTTCTTTAATTTTTTGCT 3rsquo


AGGCTTAATGTCTCTTTCCAAATAT 3rsquo

5rsquoGGGGACCACTTTGTACAAGAAAGCTG

GGTATACGCTTTGTTCGTTTGGATTAT 3rsquo


PCR Reaction

Ingredient TaqPfu Reaction Phusion Flash Master Mix

Template DNA 1 microL 1 microL

Forward Primer 1 microL 1 microL

Reverse Primer 1 microL 1 microL

10 mM dNTPs 1 microL NA

Buffer (-MgSO4) 5 microL 25 microL

MgSO4 3 microL NA

Taq Polymerase 05 microL NA

Pfu Polymerase 05 microL NA

ddH2O 37 microL 22 microL

Total Reaction Volume 50 microL 50 microL


TaqPfu Reaction Phusion Flash Master Mix

Step Temperature (degC) Time (min) Temperature (degC) Time (min)

Initial Denature 95 5 98 5

Denature 95 2 98 075

Annealing Primer Dependent 1 Primer Dependent 1

Extension 72 5 72 225

Final Extension 72 55 72 25

Cycles 35 35

103

Appendix III ndash Sequences of Pdr12p Identified Interactors


Gene Name Residues Sequence

COS8 222-381 LPKEAYRFKLTWILKRIFNLRCLPLFLYYFLIVYTSGNADLISRFLFPV

VMFFIMTRDFQNMRMIVLSVKMEHKMQFLSTIINEQESGANGWDEI

AKKMNRYLFEKKVWNNEEFFYDGLDCEWFFRRFFYRLLSLKKPMW

FASLNVELWPYIKEAQSARNEKPLK

GGT1 1-230 MSLPIIKVHWLDHSRAFRLLWLLDHLNLEYEIVPYKRDANFRAPPEL

KKIHPLGRSPLLEVQDRETGKKKILAESGFIFQYVLQHFDHSHVLMS

EDADIADQINYYLFYVEGSLQPPLMIEFILSKVKDSGMPFPISYLARK

VADKISQAYSSGEVKNQFDFVEGEISKNNGYLVDGKLSGADILMSFP

LQMAFERKFAAPEDYPAISKWLKTITSEESYAASKEKARAL

SOD1 NA LYFRYHRHVKSKIQDKEGIPGGPYPYDVPDYAGSKQWYQRRVAITA

GRKDGRKWCGQGLLQGLFDQAYRSYLRCRQKRRYPRRPRLRGH

RIFEDWCRSKTSLWCHWSNQLMLMIIYLNKNRMVSSKRINSFILK

KKKKKKKKHVGRLGLSRIRYQAYRYR

TMA7 6-64 GGKMKPLKQKKKQQQDLDPEDIAFKEKQKADAAAKKALMANMKS

GKPLVGGGIKKSGKK

TUB2 295-414 DAKNMMAAADPRNGRYLTVAAFFRGKVSVKEVEDEMHKVQSKNS

DYFVEWIPNNVQTAVCSVAPQGLDMAATFIANSTSIQELFKRVGDQF

SAMFKRKAFLHWYTSEGMDELEFSEAESN

YBR056W 395-479 QKGNLPKRPHGDDLQVDKKKIDSIIHEHEAYWNGKGKNFEHWRFED

GIKTAVDDIIAFRKFDNSLIGRWHSWKSQRRAEYVSAKK

YCK2 12-28 NSGLAVNNNTMNSQMPN

YLR154C-G NA GSSIHRHVKSKIQDKEGIPGGSTMSGHAYPYDVPDYAHGGPVEVSDE

ATVRSGRTASSADLGGSSKYSNENFEDSGERFHVNSSWTWVSRS

EMGKLRFKGLILCRPPSKGNPVKIPEPGYGFFTVTLNVETSARALGG

VIFSSQLITPELVYPEMGSYGWK

YMR315W-A 20-35 FTALRACPLRPKSLIA

ZEO1 1-109 MSEIQNKAETAAQDVQQKLEETKESLQNKGQEVKEQAEASIDNLKN

EATPEAEQVKKEEQNIADGVEQKKTEAANKVEETKKQASAAVSEKK

ETKKEGGFLKKLNRKIA

() Denotes iMYTH identified translated sequences not aligned to OFR of gene

104

Appendix IV ndash Pdr12-CT Bait Dependency Test

105

106

107

108

109

Figure 20 Pdr12p Bait Dependency Test Positive (OstI and Fur4) and negative (OstG and FurG)

control plasmids are shown in the top most panel Potential interactor proteins are listed along the left hand

side in alphabetical order SD-W is selective for the presence of prey plasmid but not interaction while

SD-WH + X-gal is selective for interaction between bait and prey Growth on medium selective for

interaction using the artificial bait strain is scored as a false positive as is failure to detect growth using the

original bait strain Both growth and blue colour are criteria used to evaluate interactions which are

genuine and specific and these are indicated by yellow stars The results of this test were used to generate

the Pdr12p interactome

110

Appendix V ndash Sequences of Ste6p Identified Interactors



VPS9 321-451 EAYQRNLKQLAEEKEEEEKKKQLEVPDELQPNGTLLKPLDEVTNIVI

SKFNELFSPIGEPTQEEALKSEQSNKEEDVSSLIKKIEENERKDTLNTL

QNMFPDMDPSLIEDVCIAKKSRIGPCVDALLSLSE

YGL081W 248-320 EEKEEEEEKEEGDDEEGEIELEIIRVKRIKGRTKIKKTLTCFSKNKKIIT

PQHSNSMWLLLIVILIFDRLLSN

111

Appendix VI ndash Ste6-CT Bait Dependency Test

Figure 21 Ste6p Bait Dependency Test Positive (OstI) and negative (OstG) control plasmids are shown

in the top panels Potential interactor proteins are listed along the left hand side SD-W is selective for the

presence of prey plasmid but not interaction while SD-WH is selective for interaction between bait and

prey Growth on medium selective for interaction using the artificial bait strain is scored as a false positive

as is failure to detect growth using the original bait strain Yellow stars indicate interactions which appear

genuine and specific The results of this test were used to generate the Ste6p interactome

112

Appendix VII ndash Pdr12 and Ste6p iMYTH Identified Interactors

Table 10 Description of Pdr12p Interactors According to the Saccharomyces Genome

Database

Gene Name Systematic Name Description

COS8 YHL048W

Nuclear membrane protein member of the DUP380 subfamily of

conserved often subtelomerically-encoded proteins regulation

suggests a potential role in the unfolded protein response

GTT1 YIR038C

ER associated glutathione S-transferase capable of

homodimerization expression induced during the diauxic shift and

throughout stationary phase functional overlap with Gtt2p Grx1p

and Grx2p

PDR5 YOR153W

Plasma membrane ATP-binding cassette (ABC) transporter

multidrug transporter actively regulated by Pdr1p also involved in

steroid transport cation resistance and cellular detoxification

during exponential growth

PDR10 YOR328W

ATP-binding cassette (ABC) transporter multidrug transporter

involved in the pleiotropic drug resistance network regulated by

Pdr1p and Pdr3p

PDR11 YIL013C


involved in multiple drug resistance mediates sterol uptake when

sterol biosynthesis is compromisedregulated by Pdr1p required for

anaerobic growth

SOD1 YJR104C

Cytosolic copper-zinc superoxide dismutase some mutations are

analogous to those that cause ALS (amyotrophic lateral sclerosis) in

humans

TMA7 YLR262C-A

Protein of unknown function that associates with ribosomes null

mutant exhibits translation defects altered polyribosome profiles

and resistance to the translation inhibitor anisomcyin

TUB2 YFL037W Beta-tubulin associates with alpha-tubulin (Tub1p and Tub3p) to

form tubulin dimer which polymerizes to form microtubules

YBR056W YBR056W Putative cytoplasmic protein of unknown function

YCK2 YNL154C

Palmitoylated plasma membrane-bound casein kinase I isoform

shares redundant functions with Yck1p in morphogenesis proper

septin assembly endocytic trafficking provides an essential

function overlapping with that of Yck1p

YLR154C-G YLR154C-G

Putative protein of unknown function identified by fungal homology

comparisons and RT-PCR this ORF is contained within RDN25-2

and RDN37-2

YMR315W-A YMR315W-A Putative protein of unknown function

ZEO1 YOL109W

Peripheral membrane protein of the plasma membrane that interacts

with Mid2p regulates the cell integrity pathway mediated by Pkc1p

and Slt2p the authentic protein is detected in a phosphorylated state

in highly purified mitochondria

113

Table 11 Description of Ste6p Interactors According to the Saccharomyces Genome

Database


VPS9 YML097C

A guanine nucleotide exchange factor involved in vesicle-mediated

vacuolar protein transport specifically stimulates the intrinsic

guanine nucleotide exchange activity of Vps21pRab5 similar to

mammalian ras inhibitors binds ubiquitin

YGL081W YGL081W Putative protein of unknown function non-essential gene interacts

genetically with CHS5 a gene involved in chitin biosynthesis

114

Appendix VIII ndash Weak Acid TECAN Assay Replicate

115

Figure 22 Sorbic and benzoic acid TECAN replicate Concentrations of acid used are indicated in the

top left hand corner of each graph YPAD is rich medium and contains no sorbic acid The legend is

found along the top and shows the strains used (A) Sorbic acid assay As the concentration of sorbic acid

increases the pdr10Δkan pdr12Δnat mutant is able to grow implying resistance even though its growth

is comparable to that of the WT strain All strains tested in this replicate are unable to grow at 10 mM

which is unexpected as growth was observed at this concentration previously (B) Benzoic acid assay

Same trends as observed with the sorbic acid assay in (A) though the double deletion mutant is able to

grow at 10 mM

ii

Mapping the Interactome of Saccharomyces cerevisiae ABC Transporters Pdr12p

and Ste6p

Dunja Damjanovic

Master of Science

Department of Molecular Genetics


2010

ABSTRACT

The ATP binding cassette (ABC) transporters represent the largest family of

transmembrane proteins and play important roles in human inherited disease such as the

multi-organ disease cystic fibrosis and cholesterol transport disorder Tangierrsquos disease

These proteins are also implicated in conferring multidrug resistance rendering many

cancer therapies ineffective as well as contributing to the pathogenicity of some

organisms The yeast ABC proteins Pdr12p a weak acid efflux pump and Ste6p the a-

factor exporter were screened for interacting partners using the integrated membrane

yeast two-hybrid (iMYTH) system to gain further insight into their biological function

Two interactors were identified for Ste6p however the Pdr12p screen identified 13 novel

interactions most notable of which are three other ABC transporters Pdr5p Pdr10p and

Pdr11p Subsequent functional analysis of double deletion mutants supports a genetic

interaction between Pdr12p and Pdr10p as the pdr12Δ pdr10Δ strain showed resistance to

increasing concentrations of weak organic acids

iii

ACKNOWLEDGMENTS





me never to give up

































Dunja Damjanovic

iv



ndash Dodie Smith



brother Srdjan

v

TABLE OF CONTENTS

ABSTRACT ii

ACKNOWLEDGMENTS iii

LIST OF TABLES viii

LIST OF FIGURES ix

APPENDICES x

ABBREVIATIONS xi

INTRODUCTION 1







17 Yeast Pdr12p 10




18 Yeast Ste6p 13
















vi



25 NubGNubI Test 27

















2101 Spot Assays 36





RESULTS 40










vii





371 Spot Assays 53



381 Spot Assays 58





DISCUSSION 65

41 GO Analysis 66












REFERENCES 84

APPENDIX 91

viii

LIST OF TABLES












ix

LIST OF FIGURES























x

APPENDICES









xi

ABBREVIATIONS











Kan ndash Kanamycin


















WT ndash Wildtype



CHAPTER 1

INTRODUCTION

2






















3






















4













homologues (8)












5














6




















7
























8

























9

























10









17 Yeast Pdr12p
















11






(24 36)


















12























CFTR (38)

13

18 Yeast Ste6p
























14
















15

























16

























17









58)















18

























19

























20

























21

























22



















23











(74-76)













24




















25

CHAPTER 2


26

























27

25 NubGNubI Test





















assessed

28



















for 2-3 days






29

























30







27 The iMYTH Assay


















31

























32

























33

















of bait and prey








34

























35















minutes

292 Gateway Cloning









36
















2101 Spot Assays









37

























38












PAGE analysis













39























40

CHAPTER 3

RESULTS

41




















42




















43








data)











deletion of PDR12

44




















45




















peptides

46




















47

















48
























49












respectively




left hand side

50























51












complex












52































53


371 Spot Assays
















54



















55
















investigation

56
















57
















58


381 Spot Assays























59

















60

















61






prolonged lag phase










62

















63



















64







65

CHAPTER 4

DISCUSSION

66

41 GO Analysis
























67























68
























69


below






















needed

70
























71


signalling pathway























72

























73
























74

























75















detected










76














77

CHAPTER 5


78

























79























80
























81























82












protein Pdr5p












83



84

REFERENCES




Cell Biol 8 67-113








577-593



Enzymol 350 87-96














584-593















85



Commun 356 1-5








J Biol Chem 279 27855-27860










17 4257-4265



65-69



18 3981-3989



2135-2138



Biol 331 155-165














86
















3094-3107











29007-29011



400-404







2784






3973-3984




87


Microbiol 3 573-581

























635









623











88






428











2005 pl3









32 349-362




Leukoc Biol 1-9

















89












































269-274



90


Chem 277 32466-32472


















91

APPENDIX

92






Toronto Toronto)





Toronto Toronto)






(94)

















DSB




R




93



Invitrogen



Recipes

05M EDTA pH 80




09 NaCl


room temperature

1M 3-AT Solution



1M Lithium Acetate



1M Tris pH 75



room temperature




10x Drop-out Mix




94

Amino Acid 2L (mg)

Isoleucine 600

Valine 3000

Adenine (A) 800

Histidine (H) 400

Leucine 2000

Lysine 600

Methionine 3000

Phenylalanine 1000

Threonine 4000

Tryptophan (W) 800

Tyrosine 600

Uracil 400

Arginine 400







20degC







20degC











95






X-Gal Solution


























periods of time








96




Agarose Gel




1M Sorbitol



Solution A






needed

Lysis Solution



Terrific Broth (TB)






T-B Buffer




Sporulation Medium




97

SD Minimal Plates








Store at 4degC





Store at 4degC












Medium













98












10 APS Solution


10X TBS pH 75



1X TBST Solution








antibody incubation

Antibodies











99




ORF Forward Reverse










ORF Forward Reverse





















100








































Gap Repair Primers







101

PDR5






GAGAGTTTACCG 3rsquo







PDR10









TGTTCGTTTGG 3rsquo

PDR11






Sequencing Primers




















102
















PCR Reaction







MgSO4 3 microL NA













Cycles 35 35

103


















GKPLVGGGIKKSGKK














ETKKEGGFLKKLNRKIA


104


105

106

107

108

109









110









111








112



Database


COS8 YHL048W




GTT1 YIR038C




and Grx2p

PDR5 YOR153W





PDR10 YOR328W



Pdr1p and Pdr3p

PDR11 YIL013C




anaerobic growth

SOD1 YJR104C



humans

TMA7 YLR262C-A







YCK2 YNL154C





YLR154C-G YLR154C-G



and RDN37-2


ZEO1 YOL109W





113


Database


VPS9 YML097C







114


115








grow at 10 mM

iii

ACKNOWLEDGMENTS





me never to give up

































Dunja Damjanovic

iv



ndash Dodie Smith



brother Srdjan

v

TABLE OF CONTENTS

ABSTRACT ii

ACKNOWLEDGMENTS iii

LIST OF TABLES viii

LIST OF FIGURES ix

APPENDICES x

ABBREVIATIONS xi

INTRODUCTION 1







17 Yeast Pdr12p 10




18 Yeast Ste6p 13
















vi



25 NubGNubI Test 27

















2101 Spot Assays 36





RESULTS 40










vii





371 Spot Assays 53



381 Spot Assays 58





DISCUSSION 65

41 GO Analysis 66












REFERENCES 84

APPENDIX 91

viii

LIST OF TABLES












ix

LIST OF FIGURES























x

APPENDICES









xi

ABBREVIATIONS











Kan ndash Kanamycin


















WT ndash Wildtype



CHAPTER 1

INTRODUCTION

2






















3






















4













homologues (8)












5














6




















7
























8

























9

























10









17 Yeast Pdr12p
















11






(24 36)


















12























CFTR (38)

13

18 Yeast Ste6p
























14
















15

























16

























17









58)















18

























19

























20

























21

























22



















23











(74-76)













24




















25

CHAPTER 2


26

























27

25 NubGNubI Test





















assessed

28



















for 2-3 days






29

























30







27 The iMYTH Assay


















31

























32

























33

















of bait and prey








34

























35















minutes

292 Gateway Cloning









36
















2101 Spot Assays









37

























38












PAGE analysis













39























40

CHAPTER 3

RESULTS

41




















42




















43








data)











deletion of PDR12

44




















45




















peptides

46




















47

















48
























49












respectively




left hand side

50























51












complex












52































53


371 Spot Assays
















54



















55
















investigation

56
















57
















58


381 Spot Assays























59

















60

















61






prolonged lag phase










62

















63



















64







65

CHAPTER 4

DISCUSSION

66

41 GO Analysis
























67























68
























69


below






















needed

70
























71


signalling pathway























72

























73
























74

























75















detected










76














77

CHAPTER 5


78

























79























80
























81























82












protein Pdr5p












83



84

REFERENCES




Cell Biol 8 67-113








577-593



Enzymol 350 87-96














584-593















85



Commun 356 1-5








J Biol Chem 279 27855-27860










17 4257-4265



65-69



18 3981-3989



2135-2138



Biol 331 155-165














86
















3094-3107











29007-29011



400-404







2784






3973-3984




87


Microbiol 3 573-581

























635









623











88






428











2005 pl3









32 349-362




Leukoc Biol 1-9

















89












































269-274



90


Chem 277 32466-32472


















91

APPENDIX

92






Toronto Toronto)





Toronto Toronto)






(94)

















DSB




R




93



Invitrogen



Recipes

05M EDTA pH 80




09 NaCl


room temperature

1M 3-AT Solution



1M Lithium Acetate



1M Tris pH 75



room temperature




10x Drop-out Mix




94

Amino Acid 2L (mg)

Isoleucine 600

Valine 3000

Adenine (A) 800

Histidine (H) 400

Leucine 2000

Lysine 600

Methionine 3000

Phenylalanine 1000

Threonine 4000

Tryptophan (W) 800

Tyrosine 600

Uracil 400

Arginine 400







20degC







20degC











95






X-Gal Solution


























periods of time








96




Agarose Gel




1M Sorbitol



Solution A






needed

Lysis Solution



Terrific Broth (TB)






T-B Buffer




Sporulation Medium




97

SD Minimal Plates








Store at 4degC





Store at 4degC












Medium













98












10 APS Solution


10X TBS pH 75



1X TBST Solution








antibody incubation

Antibodies











99




ORF Forward Reverse










ORF Forward Reverse





















100








































Gap Repair Primers







101

PDR5






GAGAGTTTACCG 3rsquo







PDR10









TGTTCGTTTGG 3rsquo

PDR11






Sequencing Primers




















102
















PCR Reaction







MgSO4 3 microL NA













Cycles 35 35

103


















GKPLVGGGIKKSGKK














ETKKEGGFLKKLNRKIA


104


105

106

107

108

109









110









111








112



Database


COS8 YHL048W




GTT1 YIR038C




and Grx2p

PDR5 YOR153W





PDR10 YOR328W



Pdr1p and Pdr3p

PDR11 YIL013C




anaerobic growth

SOD1 YJR104C



humans

TMA7 YLR262C-A







YCK2 YNL154C





YLR154C-G YLR154C-G



and RDN37-2


ZEO1 YOL109W





113


Database


VPS9 YML097C







114


115








grow at 10 mM

iv



ndash Dodie Smith



brother Srdjan

v

TABLE OF CONTENTS

ABSTRACT ii

ACKNOWLEDGMENTS iii

LIST OF TABLES viii

LIST OF FIGURES ix

APPENDICES x

ABBREVIATIONS xi

INTRODUCTION 1







17 Yeast Pdr12p 10




18 Yeast Ste6p 13
















vi



25 NubGNubI Test 27

















2101 Spot Assays 36





RESULTS 40










vii





371 Spot Assays 53



381 Spot Assays 58





DISCUSSION 65

41 GO Analysis 66












REFERENCES 84

APPENDIX 91

viii

LIST OF TABLES












ix

LIST OF FIGURES























x

APPENDICES









xi

ABBREVIATIONS











Kan ndash Kanamycin


















WT ndash Wildtype



CHAPTER 1

INTRODUCTION

2






















3






















4













homologues (8)












5














6




















7
























8

























9

























10









17 Yeast Pdr12p
















11






(24 36)


















12























CFTR (38)

13

18 Yeast Ste6p
























14
















15

























16

























17









58)















18

























19

























20

























21

























22



















23











(74-76)













24




















25

CHAPTER 2


26

























27

25 NubGNubI Test





















assessed

28



















for 2-3 days






29

























30







27 The iMYTH Assay


















31

























32

























33

















of bait and prey








34

























35















minutes

292 Gateway Cloning









36
















2101 Spot Assays









37

























38












PAGE analysis













39























40

CHAPTER 3

RESULTS

41




















42




















43








data)











deletion of PDR12

44




















45




















peptides

46




















47

















48
























49












respectively




left hand side

50























51












complex












52































53


371 Spot Assays
















54



















55
















investigation

56
















57
















58


381 Spot Assays























59

















60

















61






prolonged lag phase










62

















63



















64







65

CHAPTER 4

DISCUSSION

66

41 GO Analysis
























67























68
























69


below






















needed

70
























71


signalling pathway























72

























73
























74

























75















detected










76














77

CHAPTER 5


78

























79























80
























81























82












protein Pdr5p












83



84

REFERENCES




Cell Biol 8 67-113








577-593



Enzymol 350 87-96














584-593















85



Commun 356 1-5








J Biol Chem 279 27855-27860










17 4257-4265



65-69



18 3981-3989



2135-2138



Biol 331 155-165














86
















3094-3107











29007-29011



400-404







2784






3973-3984




87


Microbiol 3 573-581

























635









623











88






428











2005 pl3









32 349-362




Leukoc Biol 1-9

















89












































269-274



90


Chem 277 32466-32472


















91

APPENDIX

92






Toronto Toronto)





Toronto Toronto)






(94)

















DSB




R




93



Invitrogen



Recipes

05M EDTA pH 80




09 NaCl


room temperature

1M 3-AT Solution



1M Lithium Acetate



1M Tris pH 75



room temperature




10x Drop-out Mix




94

Amino Acid 2L (mg)

Isoleucine 600

Valine 3000

Adenine (A) 800

Histidine (H) 400

Leucine 2000

Lysine 600

Methionine 3000

Phenylalanine 1000

Threonine 4000

Tryptophan (W) 800

Tyrosine 600

Uracil 400

Arginine 400







20degC







20degC











95






X-Gal Solution


























periods of time








96




Agarose Gel




1M Sorbitol



Solution A






needed

Lysis Solution



Terrific Broth (TB)






T-B Buffer




Sporulation Medium




97

SD Minimal Plates








Store at 4degC





Store at 4degC












Medium













98












10 APS Solution


10X TBS pH 75



1X TBST Solution








antibody incubation

Antibodies











99




ORF Forward Reverse










ORF Forward Reverse





















100








































Gap Repair Primers







101

PDR5






GAGAGTTTACCG 3rsquo







PDR10









TGTTCGTTTGG 3rsquo

PDR11






Sequencing Primers




















102
















PCR Reaction







MgSO4 3 microL NA













Cycles 35 35

103


















GKPLVGGGIKKSGKK














ETKKEGGFLKKLNRKIA


104


105

106

107

108

109









110









111








112



Database


COS8 YHL048W




GTT1 YIR038C




and Grx2p

PDR5 YOR153W





PDR10 YOR328W



Pdr1p and Pdr3p

PDR11 YIL013C




anaerobic growth

SOD1 YJR104C



humans

TMA7 YLR262C-A







YCK2 YNL154C





YLR154C-G YLR154C-G



and RDN37-2


ZEO1 YOL109W





113


Database


VPS9 YML097C







114


115








grow at 10 mM

v

TABLE OF CONTENTS

ABSTRACT ii

ACKNOWLEDGMENTS iii

LIST OF TABLES viii

LIST OF FIGURES ix

APPENDICES x

ABBREVIATIONS xi

INTRODUCTION 1







17 Yeast Pdr12p 10




18 Yeast Ste6p 13
















vi



25 NubGNubI Test 27

















2101 Spot Assays 36





RESULTS 40










vii





371 Spot Assays 53



381 Spot Assays 58





DISCUSSION 65

41 GO Analysis 66












REFERENCES 84

APPENDIX 91

viii

LIST OF TABLES












ix

LIST OF FIGURES























x

APPENDICES









xi

ABBREVIATIONS











Kan ndash Kanamycin


















WT ndash Wildtype



CHAPTER 1

INTRODUCTION

2






















3






















4













homologues (8)












5














6




















7
























8

























9

























10









17 Yeast Pdr12p
















11






(24 36)


















12























CFTR (38)

13

18 Yeast Ste6p
























14
















15

























16

























17









58)















18

























19

























20

























21

























22



















23











(74-76)













24




















25

CHAPTER 2


26

























27

25 NubGNubI Test





















assessed

28



















for 2-3 days






29

























30







27 The iMYTH Assay


















31

























32

























33

















of bait and prey








34

























35















minutes

292 Gateway Cloning









36
















2101 Spot Assays









37

























38












PAGE analysis













39























40

CHAPTER 3

RESULTS

41




















42




















43








data)











deletion of PDR12

44




















45




















peptides

46




















47

















48
























49












respectively




left hand side

50























51












complex












52































53


371 Spot Assays
















54



















55
















investigation

56
















57
















58


381 Spot Assays























59

















60

















61






prolonged lag phase










62

















63



















64







65

CHAPTER 4

DISCUSSION

66

41 GO Analysis
























67























68
























69


below






















needed

70
























71


signalling pathway























72

























73
























74

























75















detected










76














77

CHAPTER 5


78

























79























80
























81























82












protein Pdr5p












83



84

REFERENCES




Cell Biol 8 67-113








577-593



Enzymol 350 87-96














584-593















85



Commun 356 1-5








J Biol Chem 279 27855-27860










17 4257-4265



65-69



18 3981-3989



2135-2138



Biol 331 155-165














86
















3094-3107











29007-29011



400-404







2784






3973-3984




87


Microbiol 3 573-581

























635









623











88






428











2005 pl3









32 349-362




Leukoc Biol 1-9

















89












































269-274



90


Chem 277 32466-32472


















91

APPENDIX

92






Toronto Toronto)





Toronto Toronto)






(94)

















DSB




R




93



Invitrogen



Recipes

05M EDTA pH 80




09 NaCl


room temperature

1M 3-AT Solution



1M Lithium Acetate



1M Tris pH 75



room temperature




10x Drop-out Mix




94

Amino Acid 2L (mg)

Isoleucine 600

Valine 3000

Adenine (A) 800

Histidine (H) 400

Leucine 2000

Lysine 600

Methionine 3000

Phenylalanine 1000

Threonine 4000

Tryptophan (W) 800

Tyrosine 600

Uracil 400

Arginine 400







20degC







20degC











95






X-Gal Solution


























periods of time








96




Agarose Gel




1M Sorbitol



Solution A






needed

Lysis Solution



Terrific Broth (TB)






T-B Buffer




Sporulation Medium




97

SD Minimal Plates








Store at 4degC





Store at 4degC












Medium













98












10 APS Solution


10X TBS pH 75



1X TBST Solution








antibody incubation

Antibodies











99




ORF Forward Reverse










ORF Forward Reverse





















100








































Gap Repair Primers







101

PDR5






GAGAGTTTACCG 3rsquo







PDR10









TGTTCGTTTGG 3rsquo

PDR11






Sequencing Primers




















102
















PCR Reaction







MgSO4 3 microL NA













Cycles 35 35

103


















GKPLVGGGIKKSGKK














ETKKEGGFLKKLNRKIA


104


105

106

107

108

109









110









111








112



Database


COS8 YHL048W




GTT1 YIR038C




and Grx2p

PDR5 YOR153W





PDR10 YOR328W



Pdr1p and Pdr3p

PDR11 YIL013C




anaerobic growth

SOD1 YJR104C



humans

TMA7 YLR262C-A







YCK2 YNL154C





YLR154C-G YLR154C-G



and RDN37-2


ZEO1 YOL109W





113


Database


VPS9 YML097C







114


115








grow at 10 mM

vi



25 NubGNubI Test 27

















2101 Spot Assays 36





RESULTS 40










vii





371 Spot Assays 53



381 Spot Assays 58





DISCUSSION 65

41 GO Analysis 66












REFERENCES 84

APPENDIX 91

viii

LIST OF TABLES












ix

LIST OF FIGURES























x

APPENDICES









xi

ABBREVIATIONS











Kan ndash Kanamycin


















WT ndash Wildtype



CHAPTER 1

INTRODUCTION

2






















3






















4













homologues (8)












5














6




















7
























8

























9

























10









17 Yeast Pdr12p
















11






(24 36)


















12























CFTR (38)

13

18 Yeast Ste6p
























14
















15

























16

























17









58)















18

























19

























20

























21

























22



















23











(74-76)













24




















25

CHAPTER 2


26

























27

25 NubGNubI Test





















assessed

28



















for 2-3 days






29

























30







27 The iMYTH Assay


















31

























32

























33

















of bait and prey








34

























35















minutes

292 Gateway Cloning









36
















2101 Spot Assays









37

























38












PAGE analysis













39























40

CHAPTER 3

RESULTS

41




















42




















43








data)











deletion of PDR12

44




















45




















peptides

46




















47

















48
























49












respectively




left hand side

50























51












complex












52































53


371 Spot Assays
















54



















55
















investigation

56
















57
















58


381 Spot Assays























59

















60

















61






prolonged lag phase










62

















63



















64







65

CHAPTER 4

DISCUSSION

66

41 GO Analysis
























67























68
























69


below






















needed

70
























71


signalling pathway























72

























73
























74

























75















detected










76














77

CHAPTER 5


78

























79























80
























81























82












protein Pdr5p












83



84

REFERENCES




Cell Biol 8 67-113








577-593



Enzymol 350 87-96














584-593















85



Commun 356 1-5








J Biol Chem 279 27855-27860










17 4257-4265



65-69



18 3981-3989



2135-2138



Biol 331 155-165














86
















3094-3107











29007-29011



400-404







2784






3973-3984




87


Microbiol 3 573-581

























635









623











88






428











2005 pl3









32 349-362




Leukoc Biol 1-9

















89












































269-274



90


Chem 277 32466-32472


















91

APPENDIX

92






Toronto Toronto)





Toronto Toronto)






(94)

















DSB




R




93



Invitrogen



Recipes

05M EDTA pH 80




09 NaCl


room temperature

1M 3-AT Solution



1M Lithium Acetate



1M Tris pH 75



room temperature




10x Drop-out Mix




94

Amino Acid 2L (mg)

Isoleucine 600

Valine 3000

Adenine (A) 800

Histidine (H) 400

Leucine 2000

Lysine 600

Methionine 3000

Phenylalanine 1000

Threonine 4000

Tryptophan (W) 800

Tyrosine 600

Uracil 400

Arginine 400







20degC







20degC











95






X-Gal Solution


























periods of time








96




Agarose Gel




1M Sorbitol



Solution A






needed

Lysis Solution



Terrific Broth (TB)






T-B Buffer




Sporulation Medium




97

SD Minimal Plates








Store at 4degC





Store at 4degC












Medium













98












10 APS Solution


10X TBS pH 75



1X TBST Solution








antibody incubation

Antibodies











99




ORF Forward Reverse










ORF Forward Reverse





















100








































Gap Repair Primers







101

PDR5






GAGAGTTTACCG 3rsquo







PDR10









TGTTCGTTTGG 3rsquo

PDR11






Sequencing Primers




















102
















PCR Reaction







MgSO4 3 microL NA













Cycles 35 35

103


















GKPLVGGGIKKSGKK














ETKKEGGFLKKLNRKIA


104


105

106

107

108

109









110









111








112



Database


COS8 YHL048W




GTT1 YIR038C




and Grx2p

PDR5 YOR153W





PDR10 YOR328W



Pdr1p and Pdr3p

PDR11 YIL013C




anaerobic growth

SOD1 YJR104C



humans

TMA7 YLR262C-A







YCK2 YNL154C





YLR154C-G YLR154C-G



and RDN37-2


ZEO1 YOL109W





113


Database


VPS9 YML097C







114


115








grow at 10 mM

vii





371 Spot Assays 53



381 Spot Assays 58





DISCUSSION 65

41 GO Analysis 66












REFERENCES 84

APPENDIX 91

viii

LIST OF TABLES












ix

LIST OF FIGURES























x

APPENDICES









xi

ABBREVIATIONS











Kan ndash Kanamycin


















WT ndash Wildtype



CHAPTER 1

INTRODUCTION

2






















3






















4













homologues (8)












5














6




















7
























8

























9

























10









17 Yeast Pdr12p
















11






(24 36)


















12























CFTR (38)

13

18 Yeast Ste6p
























14
















15

























16

























17









58)















18

























19

























20

























21

























22



















23











(74-76)













24




















25

CHAPTER 2


26

























27

25 NubGNubI Test





















assessed

28



















for 2-3 days






29

























30







27 The iMYTH Assay


















31

























32

























33

















of bait and prey








34

























35















minutes

292 Gateway Cloning









36
















2101 Spot Assays









37

























38












PAGE analysis













39























40

CHAPTER 3

RESULTS

41




















42




















43








data)











deletion of PDR12

44




















45




















peptides

46




















47

















48
























49












respectively




left hand side

50























51












complex












52































53


371 Spot Assays
















54



















55
















investigation

56
















57
















58


381 Spot Assays























59

















60

















61






prolonged lag phase










62

















63



















64







65

CHAPTER 4

DISCUSSION

66

41 GO Analysis
























67























68
























69


below






















needed

70
























71


signalling pathway























72

























73
























74

























75















detected










76














77

CHAPTER 5


78

























79























80
























81























82












protein Pdr5p












83



84

REFERENCES




Cell Biol 8 67-113








577-593



Enzymol 350 87-96














584-593















85



Commun 356 1-5








J Biol Chem 279 27855-27860










17 4257-4265



65-69



18 3981-3989



2135-2138



Biol 331 155-165














86
















3094-3107











29007-29011



400-404







2784






3973-3984




87


Microbiol 3 573-581

























635









623











88






428











2005 pl3









32 349-362




Leukoc Biol 1-9

















89












































269-274



90


Chem 277 32466-32472


















91

APPENDIX

92






Toronto Toronto)





Toronto Toronto)






(94)

















DSB




R




93



Invitrogen



Recipes

05M EDTA pH 80




09 NaCl


room temperature

1M 3-AT Solution



1M Lithium Acetate



1M Tris pH 75



room temperature




10x Drop-out Mix




94

Amino Acid 2L (mg)

Isoleucine 600

Valine 3000

Adenine (A) 800

Histidine (H) 400

Leucine 2000

Lysine 600

Methionine 3000

Phenylalanine 1000

Threonine 4000

Tryptophan (W) 800

Tyrosine 600

Uracil 400

Arginine 400







20degC







20degC











95






X-Gal Solution


























periods of time








96




Agarose Gel




1M Sorbitol



Solution A






needed

Lysis Solution



Terrific Broth (TB)






T-B Buffer




Sporulation Medium




97

SD Minimal Plates








Store at 4degC





Store at 4degC












Medium













98












10 APS Solution


10X TBS pH 75



1X TBST Solution








antibody incubation

Antibodies











99




ORF Forward Reverse










ORF Forward Reverse





















100








































Gap Repair Primers







101

PDR5






GAGAGTTTACCG 3rsquo







PDR10









TGTTCGTTTGG 3rsquo

PDR11






Sequencing Primers




















102
















PCR Reaction







MgSO4 3 microL NA













Cycles 35 35

103


















GKPLVGGGIKKSGKK














ETKKEGGFLKKLNRKIA


104


105

106

107

108

109









110









111








112



Database


COS8 YHL048W




GTT1 YIR038C




and Grx2p

PDR5 YOR153W





PDR10 YOR328W



Pdr1p and Pdr3p

PDR11 YIL013C




anaerobic growth

SOD1 YJR104C



humans

TMA7 YLR262C-A







YCK2 YNL154C





YLR154C-G YLR154C-G



and RDN37-2


ZEO1 YOL109W





113


Database


VPS9 YML097C







114


115








grow at 10 mM

viii

LIST OF TABLES












ix

LIST OF FIGURES























x

APPENDICES









xi

ABBREVIATIONS











Kan ndash Kanamycin


















WT ndash Wildtype



CHAPTER 1

INTRODUCTION

2






















3






















4













homologues (8)












5














6




















7
























8

























9

























10









17 Yeast Pdr12p
















11






(24 36)


















12























CFTR (38)

13

18 Yeast Ste6p
























14
















15

























16

























17









58)















18

























19

























20

























21

























22



















23











(74-76)













24




















25

CHAPTER 2


26

























27

25 NubGNubI Test





















assessed

28



















for 2-3 days






29

























30







27 The iMYTH Assay


















31

























32

























33

















of bait and prey








34

























35















minutes

292 Gateway Cloning









36
















2101 Spot Assays









37

























38












PAGE analysis













39























40

CHAPTER 3

RESULTS

41




















42




















43








data)











deletion of PDR12

44




















45




















peptides

46




















47

















48
























49












respectively




left hand side

50























51












complex












52































53


371 Spot Assays
















54



















55
















investigation

56
















57
















58


381 Spot Assays























59

















60

















61






prolonged lag phase










62

















63



















64







65

CHAPTER 4

DISCUSSION

66

41 GO Analysis
























67























68
























69


below






















needed

70
























71


signalling pathway























72

























73
























74

























75















detected










76














77

CHAPTER 5


78

























79























80
























81























82












protein Pdr5p












83



84

REFERENCES




Cell Biol 8 67-113








577-593



Enzymol 350 87-96














584-593















85



Commun 356 1-5








J Biol Chem 279 27855-27860










17 4257-4265



65-69



18 3981-3989



2135-2138



Biol 331 155-165














86
















3094-3107











29007-29011



400-404







2784






3973-3984




87


Microbiol 3 573-581

























635









623











88






428











2005 pl3









32 349-362




Leukoc Biol 1-9

















89












































269-274



90


Chem 277 32466-32472


















91

APPENDIX

92






Toronto Toronto)





Toronto Toronto)






(94)

















DSB




R




93



Invitrogen



Recipes

05M EDTA pH 80




09 NaCl


room temperature

1M 3-AT Solution



1M Lithium Acetate



1M Tris pH 75



room temperature




10x Drop-out Mix




94

Amino Acid 2L (mg)

Isoleucine 600

Valine 3000

Adenine (A) 800

Histidine (H) 400

Leucine 2000

Lysine 600

Methionine 3000

Phenylalanine 1000

Threonine 4000

Tryptophan (W) 800

Tyrosine 600

Uracil 400

Arginine 400







20degC







20degC











95






X-Gal Solution


























periods of time








96




Agarose Gel




1M Sorbitol



Solution A






needed

Lysis Solution



Terrific Broth (TB)






T-B Buffer




Sporulation Medium




97

SD Minimal Plates








Store at 4degC





Store at 4degC












Medium













98












10 APS Solution


10X TBS pH 75



1X TBST Solution








antibody incubation

Antibodies











99




ORF Forward Reverse










ORF Forward Reverse





















100








































Gap Repair Primers







101

PDR5






GAGAGTTTACCG 3rsquo







PDR10









TGTTCGTTTGG 3rsquo

PDR11






Sequencing Primers




















102
















PCR Reaction







MgSO4 3 microL NA













Cycles 35 35

103


















GKPLVGGGIKKSGKK














ETKKEGGFLKKLNRKIA


104


105

106

107

108

109









110









111








112



Database


COS8 YHL048W




GTT1 YIR038C




and Grx2p

PDR5 YOR153W





PDR10 YOR328W



Pdr1p and Pdr3p

PDR11 YIL013C




anaerobic growth

SOD1 YJR104C



humans

TMA7 YLR262C-A







YCK2 YNL154C





YLR154C-G YLR154C-G



and RDN37-2


ZEO1 YOL109W





113


Database


VPS9 YML097C







114


115








grow at 10 mM

ix

LIST OF FIGURES























x

APPENDICES









xi

ABBREVIATIONS











Kan ndash Kanamycin


















WT ndash Wildtype



CHAPTER 1

INTRODUCTION

2






















3






















4













homologues (8)












5














6




















7
























8

























9

























10









17 Yeast Pdr12p
















11






(24 36)


















12























CFTR (38)

13

18 Yeast Ste6p
























14
















15

























16

























17









58)















18

























19

























20

























21

























22



















23











(74-76)













24




















25

CHAPTER 2


26

























27

25 NubGNubI Test





















assessed

28



















for 2-3 days






29

























30







27 The iMYTH Assay


















31

























32

























33

















of bait and prey








34

























35















minutes

292 Gateway Cloning









36
















2101 Spot Assays









37

























38












PAGE analysis













39























40

CHAPTER 3

RESULTS

41




















42




















43








data)











deletion of PDR12

44




















45




















peptides

46




















47

















48
























49












respectively




left hand side

50























51












complex












52































53


371 Spot Assays
















54



















55
















investigation

56
















57
















58


381 Spot Assays























59

















60

















61






prolonged lag phase










62

















63



















64







65

CHAPTER 4

DISCUSSION

66

41 GO Analysis
























67























68
























69


below






















needed

70
























71


signalling pathway























72

























73
























74

























75















detected










76














77

CHAPTER 5


78

























79























80
























81























82












protein Pdr5p












83



84

REFERENCES




Cell Biol 8 67-113








577-593



Enzymol 350 87-96














584-593















85



Commun 356 1-5








J Biol Chem 279 27855-27860










17 4257-4265



65-69



18 3981-3989



2135-2138



Biol 331 155-165














86
















3094-3107











29007-29011



400-404







2784






3973-3984




87


Microbiol 3 573-581

























635









623











88






428











2005 pl3









32 349-362




Leukoc Biol 1-9

















89












































269-274



90


Chem 277 32466-32472


















91

APPENDIX

92






Toronto Toronto)





Toronto Toronto)






(94)

















DSB




R




93



Invitrogen



Recipes

05M EDTA pH 80




09 NaCl


room temperature

1M 3-AT Solution



1M Lithium Acetate



1M Tris pH 75



room temperature




10x Drop-out Mix




94

Amino Acid 2L (mg)

Isoleucine 600

Valine 3000

Adenine (A) 800

Histidine (H) 400

Leucine 2000

Lysine 600

Methionine 3000

Phenylalanine 1000

Threonine 4000

Tryptophan (W) 800

Tyrosine 600

Uracil 400

Arginine 400







20degC







20degC











95






X-Gal Solution


























periods of time








96




Agarose Gel




1M Sorbitol



Solution A






needed

Lysis Solution



Terrific Broth (TB)






T-B Buffer




Sporulation Medium




97

SD Minimal Plates








Store at 4degC





Store at 4degC












Medium













98












10 APS Solution


10X TBS pH 75



1X TBST Solution








antibody incubation

Antibodies











99




ORF Forward Reverse










ORF Forward Reverse





















100








































Gap Repair Primers







101

PDR5






GAGAGTTTACCG 3rsquo







PDR10









TGTTCGTTTGG 3rsquo

PDR11






Sequencing Primers




















102
















PCR Reaction







MgSO4 3 microL NA













Cycles 35 35

103


















GKPLVGGGIKKSGKK














ETKKEGGFLKKLNRKIA


104


105

106

107

108

109









110









111








112



Database


COS8 YHL048W




GTT1 YIR038C




and Grx2p

PDR5 YOR153W





PDR10 YOR328W



Pdr1p and Pdr3p

PDR11 YIL013C




anaerobic growth

SOD1 YJR104C



humans

TMA7 YLR262C-A







YCK2 YNL154C





YLR154C-G YLR154C-G



and RDN37-2


ZEO1 YOL109W





113


Database


VPS9 YML097C







114


115








grow at 10 mM

x

APPENDICES









xi

ABBREVIATIONS











Kan ndash Kanamycin


















WT ndash Wildtype



CHAPTER 1

INTRODUCTION

2






















3






















4













homologues (8)












5














6




















7
























8

























9

























10









17 Yeast Pdr12p
















11






(24 36)


















12























CFTR (38)

13

18 Yeast Ste6p
























14
















15

























16

























17









58)















18

























19

























20

























21

























22



















23











(74-76)













24




















25

CHAPTER 2


26

























27

25 NubGNubI Test





















assessed

28



















for 2-3 days






29

























30







27 The iMYTH Assay


















31

























32

























33

















of bait and prey








34

























35















minutes

292 Gateway Cloning









36
















2101 Spot Assays









37

























38












PAGE analysis













39























40

CHAPTER 3

RESULTS

41




















42




















43








data)











deletion of PDR12

44




















45




















peptides

46




















47

















48
























49












respectively




left hand side

50























51












complex












52































53


371 Spot Assays
















54



















55
















investigation

56
















57
















58


381 Spot Assays























59

















60

















61






prolonged lag phase










62

















63



















64







65

CHAPTER 4

DISCUSSION

66

41 GO Analysis
























67























68
























69


below






















needed

70
























71


signalling pathway























72

























73
























74

























75















detected










76














77

CHAPTER 5


78

























79























80
























81























82












protein Pdr5p












83



84

REFERENCES




Cell Biol 8 67-113








577-593



Enzymol 350 87-96














584-593















85



Commun 356 1-5








J Biol Chem 279 27855-27860










17 4257-4265



65-69



18 3981-3989



2135-2138



Biol 331 155-165














86
















3094-3107











29007-29011



400-404







2784






3973-3984




87


Microbiol 3 573-581

























635









623











88






428











2005 pl3









32 349-362




Leukoc Biol 1-9

















89












































269-274



90


Chem 277 32466-32472


















91

APPENDIX

92






Toronto Toronto)





Toronto Toronto)






(94)

















DSB




R




93



Invitrogen



Recipes

05M EDTA pH 80




09 NaCl


room temperature

1M 3-AT Solution



1M Lithium Acetate



1M Tris pH 75



room temperature




10x Drop-out Mix




94

Amino Acid 2L (mg)

Isoleucine 600

Valine 3000

Adenine (A) 800

Histidine (H) 400

Leucine 2000

Lysine 600

Methionine 3000

Phenylalanine 1000

Threonine 4000

Tryptophan (W) 800

Tyrosine 600

Uracil 400

Arginine 400







20degC







20degC











95






X-Gal Solution


























periods of time








96




Agarose Gel




1M Sorbitol



Solution A






needed

Lysis Solution



Terrific Broth (TB)






T-B Buffer




Sporulation Medium




97

SD Minimal Plates








Store at 4degC





Store at 4degC












Medium













98












10 APS Solution


10X TBS pH 75



1X TBST Solution








antibody incubation

Antibodies











99




ORF Forward Reverse










ORF Forward Reverse





















100








































Gap Repair Primers







101

PDR5






GAGAGTTTACCG 3rsquo







PDR10









TGTTCGTTTGG 3rsquo

PDR11






Sequencing Primers




















102
















PCR Reaction







MgSO4 3 microL NA













Cycles 35 35

103


















GKPLVGGGIKKSGKK














ETKKEGGFLKKLNRKIA


104


105

106

107

108

109









110









111








112



Database


COS8 YHL048W




GTT1 YIR038C




and Grx2p

PDR5 YOR153W





PDR10 YOR328W



Pdr1p and Pdr3p

PDR11 YIL013C




anaerobic growth

SOD1 YJR104C



humans

TMA7 YLR262C-A







YCK2 YNL154C





YLR154C-G YLR154C-G



and RDN37-2


ZEO1 YOL109W





113


Database


VPS9 YML097C







114


115








grow at 10 mM

Mapping the Interactome of Saccharomyces cerevisiae ABC ......Two interactors were identified for...

Documents

Transcript of Mapping the Interactome of Saccharomyces cerevisiae ABC ......Two interactors were identified for...