Regulation of Hsp70 function by nucleotide-exchange factors919735/FULLTEXT01.pdfRegulation of Hsp70...

Regulation of Hsp70 function by nucleotide-exchange factors

Naveen Kumar Chandappa Gowda

II

©Naveen Kumar Chandappa Gowda, Stockholm University 2016 ISBN 978-91-7649-376-2 Printed by Holmbergs, Malmö 2016 Distributor: Department of Molecular Biosciences, The Wenner-Gren Institute

III

Dedicated to Sri Rudregowda S

and Smt. Shakunthala Y P

V

1. Research summary Protein folding is the process in which polypeptides in their non-native states attain the unique folds of their native states.Adverse environmental conditions and genetic predisposition challenge the folding process and accelerate the production of proteotoxic misfolded proteins. Misfolded proteins are selective-ly recognized and removed from the cell by processes of protein quality control (PQC). In PQC molecular chaperones of the Heat shock protein 70 kDa (Hsp70) family play important roles by recognizing and facilitating the removal of misfolded proteins. Hsp70 function is dependent on cofactors that regulate the intrinsic ATPase activity of the chaperone. In this thesis I have used yeast genetic, cell biological and biochemical experiments to gain insight into the regulation of Hsp70 function in PQC by nucleotide-exchange factors (NEFs). Study I shows that the NEF Fes1 is a key factor essential for cytosolic PQC. A reverse genetics approach demonstrated that Fes1 NEF activity is required for the degradation of misfolded proteins associated with Hsp70 by the ubiquitin-proteasome system. Specifically, Fes1 association with Hsp70-substrate com-plexes promotes interaction of the substrate with downstream ubiquitin E3 ligase Ubr1. The consequences of genetic removal of FES1 (fes1Δ) are the fail-ure to degrade misfolded proteins, the accumulation of protein aggregates and constitutive induction of the heat-shock response. Taken the experimental data together, Fes1 targets misfolded proteins for degradation by releasing them from Hsp70. Study II describes an unusual example of alternative splicing of FES1 transcripts that leads to the expression of the two alternative splice isoforms Fes1S and Fes1L. Both isoforms are functional NEFs but localize to different compartments. Fes1S is localized to the cytosol and is required for the efficient degradation of Hsp70-associated misfolded proteins. In contrast, Fes1L is targeted to the nucleus and represents the first identified nuclear NEF in yeast. The identification of distinctly localized Fes1 isoforms have implica-tions for the understanding of the mechanisms underlying nucleo-cytoplasmic PQC. Study III reports on the mechanism that Fes1 employs to regulate Hsp70 function. Specifically Fes1 carries an N-terminal domain (NTD) that is conserved throughout the fungal kingdom. The NTD is flexible, modular and is required for the cellular function of Fes1. Importantly, the NTD forms ATP-sensitive complexes with Hsp70 suggesting that it competes substrates of the chaperone during Fes1-Hsp70 interactions. Study IV reports on methodologi-cal development for the efficient assembly of bacterial protein-expression plasmids using yeast homologous recombination cloning and the novel vector pSUMO-YHRC. The findings support the notion that Fes1 plays a key role in determining the fate of Hsp70-associated misfolded substrates and thereby target them for proteasomal degradation. From a broader perspective, the find-ings provide information essential to develop models that describe how Hsp70 function is regulated by different NEFs to participate in protein folding and degradation.

VI

2. List of publications

1. Gowda NKC, Kandasamy G, Froehlich MS, Dohmen RJ, Andréasson C. Hsp70 nucleotide exchange factor Fes1 is essential for ubiquitin-dependent degradation of misfolded cytosolic proteins. Proc Natl Acad Sci U S A. 2013 Apr 9; 110(15): 5975-80.

2. Gowda NKC, Kaimal JM, Masser A, Kang W, Friedlander MR,

Andréasson C. Cytosolic splice isoform of Hsp70 nucleotide exchange factor Fes1 is required for the degradation of misfolded proteins in yeast, Mol Biol Cell. 2016 Apr 15; 27(8): 1210-1219.

3. Gowda NKC, Kaimal JM, Liebau J, Andréasson C. The N-terminal

domain of nucleotide exchange factor Fes1 interacts with Hsp70 and is critical for cellular function. Manuscript 2016.

Method development included in the thesis 4. Holmberg MA, Gowda NKC, Andréasson C. A versatile

bacterial expression vector designed for single-step cloning of multiple DNA fragments using homologous recombination. Protein Expr Purif. 2014 Jun; 98: 38-45.

VII

3. Abbreviations

AAA+ ADP ATP BAG CTD DNA DnaJ DUBs ERAD Fes1 FFL Hsf1 Hsp HspBP1 HSRE IPOD INQ JUNQ NBD NEF NLS NTD PQC RAC RNA San1 SBD Sis1 Snl1 Ssa Ssb Sse1 Uba Ubc Ubr1 UPS Ydj1

- ATPases associated with various cellular activities - Adenosine diphosphate - Adenosine triphosphate - Bcl2-associated anthanogene - C-terminal domain - Deoxyribonucleic acid - DNA binding domain J protein - Deubiquitinating enzymes - Endoplasmic reticulum associated degradation - Factor exchange for Ssa1 protein - Firefly luciferase - Heat shock factor 1 - Heat shock protein - Heat shock protein binding protein 1 - Heat shock response element - Insoluble protein deposit - Intranuclear quality control - Juxtanuclear quality control - Nucleotide binding domain - Nucleotide exchange factor - Nuclear localization signal - N-terminal domain - Protein quality control - Ribosome associated complex - Ribonucleic acid - Sir protein antagonist - Substrate binding domain - Slt4 suppressor - Suppressor of Nup116-C lethal - Stress seventy subfamily A - Stress seventy subfamily B - Stress seventy E1 protein - Ubiquitin activating enzyme - Ubiquitin conjugating enzyme - Ubiquitin protein ligase E3 component n-recognin1 - Ubiquitin-proteasome system - Yeast DnaJ

IX

Contents

1. Research summary………………………………………………….. V

2. List of publications…………………………………………………. VI

3. Abbreviations………………………………………………………. VII

4. Introduction………………………………………………………... 11

4.1 Protein structure……………………………………………….. 11

4.2 Protein folding…………………………………………………. 11

4.3 Protein misfolding……………………………………………... 13

4.4 The heat-shock response………………………………………. 14

4.5 Proteostasis……………………………………………………. 15

4.6 Molecular chaperones………………………………………….. 16

4.7 Hsp70 molecular chaperones…………………………………... 19

4.8 Hsp70 in yeast…………………………………………………. 20

4.9 Co-chaperones of Hsp70………………………………………. 21

4.9.1 Hsp40…………………………………………………….. 21

4.10 Nucleotide-exchange factors………………………………….. 23

4.10.1 The Hsp110 family - Sse1 and Sse2……………………… 24

4.10.2 The BAG family - Snl1…………………………………... 26

4.10.3 The HspBP1 family - Fes1………………………………. 27

4.11 The ubiquitin-proteasome system……………………………... 29

4.12 Deubiquitylation……………………………………………… 31

4.13 The 26S proteasome…………………………………………... 31

4.14 Protein quality control in the cytosol and nucleus……………... 32

4.15 Autophagy……….…………………………………………… 35

4.16 Alternative splicing in yeast…………………………………… 35

4.17 The model organism………………………………………….. 37

5. Aims………………………………………………………………... 39

5.1 Study I…………………………………………………………. 40

5.1.1 Aim……………………………………………………….. 40

5.1.2 Results and discussion…………………………………….. 40

X

5.2 Study II…………………………………………………………

44

5.2.1 Aim……………………………………………………….. 44


5.3 Study III………………………………………………………... 48

5.3.1 Aim……………………………………………………….. 48


5.4 Study IV………………………………………………………... 51

5.4.1 Aim……………………………………………………….. 51


6. Conclusions and Outlook………………………………………….... 53

7. Sammanfattning på svenska ……………………………………….... 57

8. Acknowledgments…………………………………………………... 58

9. References…………………………………………………………... 61

11

4. Introduction

4.1 Protein structure Proteins are the most abundant macromolecules in the cell and are essential for life. These polypeptides constitute the structural and enzymatic building blocks of a cell, and include such entities as antibodies, enzymes, hormones, transporters and muscle fibres. A protein molecule is a chain of amino acids, in which the neighbouring residues are linked by covalent peptide bonds. The amino acid chain is structured at four levels with increasing complexity. The primary structure defines the sequence of amino acid residues. The secondary structure defines regular structural patterns of α-helices and β-sheets. The tertiary structure describes the three-dimensional fold of the polypeptide. Finally, the quaternary structure is the arrangement of two or more polypeptides in complexes. 4.2 Protein folding Proteins reach their unique three-dimensional structures by the process of protein folding. In vitro experiments have established that the structural information required for the folding of a protein to its native conformation is genetically encoded within the primary amino acid sequence of the polypeptide (Anfinsen, 1973). However, protein folding is also highly dependent on the composition of the solvent. To be able to fold a protein to its native conformation, hydrophobic amino acids are excluded from the surrounding aqueous environment. In vitro studies have shown that proteins are thermodynamically stable when hydrophobic groups are assembled rather than extended into the aqueous environment (Anfinsen, 1973). Hence, in order to achive a stably folded protein, hydrophobic amino acids must be buried within the structure and hydrophilic residues retained on the surface. This hydrophobic collapse is a main driving force in protein folding.

12

Combining theoretical and experimental studies, protein folding can broadly be explained by three pathways, as in Figure 1 (Cattaneo et al., 2012). The first pathway is based on the concept of hydrophobic collapse, in which polar and non-polar amino acids separate within the protein, and interaction with the surrounding aqueous environment leads to the formation of a hydrophobic core. This is followed by the formation of intermediate secondary structures that further fold into the native structure (Agashe et al., 1995). The second pathway is based on a nucleation-growth mechanism that relies on the formation of local secondary structure that acts as a scaffold on which to build the native structure (Fersht and Daggett, 2002). Finally, the third pathway is a hierarchical process that functions by the formation of secondary structure elements, which is followed by tertiary structure organisation (Baldwin and Rose, 1999). Recent simulation studies have shown that multiple mechanisms are active simultaneously and that folding pathways are influenced by both sequence context and the cellular environment (Udgaonkar, 2008).

Figure 1: Mechanism of protein folding. A main event is hydrophobic collapse due to interactions between hydrophobic residues and the exclusion of water. During nucleation a scaffold is formed that facilitates further steps in the folding. Secondary structure formation is the first step in a hierarchical process that eventually results in the formation of the native structure. Adapted from (Cattaneo et al., 2012). In cells with their complex matrix of biomolecules, protein folding is not generally a spontaneous process (Hartl and Hayer-Hartl, 2002). The high levels of macromolecules and the low levels of free water are believed to challenge the folding process and make it necessary to employ cellular machineries that promote folding. Moreover, many cellular proteins are large and some are multidomain entities. These take a long time to fold with an increased risk of

Unfolded peptide Native protein

Hydrophobic collapse

Nucleation

Secondary structure formation

13

inappropriate interactions that hamper the folding process. In vitro experiments have shown that folding is dependent on the molecular size of the protein, for example single-domain proteins fold rapidly (within milliseconds) by burying exposed hydrophobic amino acids, while larger proteins take longer to fold (Dobson and Karplus, 1999). 4.3 Protein misfolding Protein misfolding is the phenomenon in which a protein becomes non-functional through acquiring a non-native conformation. Protein misfolding is energetically costly, and it decreases the concentration of the functional proteins while generating misfolded proteins that are harmful to the cell (Geiler-Samerotte et al., 2011; Stefani and Dobson, 2003). The cost of protein misfolding in the eukaryotic cell can be observed as induced transcriptional stress-response pathways and reduced growth rates (Brauer et al., 2008). The presence of higher concentrations of misfolded or unfolded proteins in the cells is the primary cause of the induction of stress-responsive gene regulatory programs, such as the heat-shock response (Parsell and Sauer, 1989). Protein misfolding is a spontaneous process and is affected by the particular protein that is to fold, and by environmental and genetic conditions. For example, larger proteins with multiple domains or with longer exposed hydrophobic stretches often fold inefficiently owing to the formation of populations of partially folded intermediates that tend to aggregate (Figure 2) (Hartl and Hayer-Hartl, 2002). Genetic mutations or polymorphisms affect the translated amino acid sequence and may increase the likelihood of protein misfolding. Other causes of protein misfolding are biosynthetic errors and the absence of necessary post-translation binding partners (McClellan et al., 2005b). Environmental conditions also affect the likelihood of protein misfolding. For example, heat stress is a well-known accelerator of protein misfolding, while nutritional stress is another. A number of experimental conditions, such as the addition of structural amino acid analogues that are incorporated during translation and hamper folding pathway, also trigger massive protein misfolding. Misfolded proteins tend to aggregate into inclusions due to interactions between exposed hydrophobic segments and β-sheet stacking. In certain cases, proteins aggregate as fibrils called ‘‘amyloids’’ (Figure 2). Typical amyloids are

14

observed as thread-like structures, and may assemble into larger aggregates or plaques. These structures are associated with diseases such as Alzheimer’s and Huntington’s disease (Dobson and Karplus, 1999; Hartl and Hayer-Hartl, 2002). Such aggregates are resistant to degradation, are toxic and cause cell death (Dobson, 2004).

Figure 2: Schematic representation of folding states of newly synthesized polypeptide chains. The monomeric states include the unfolded state (U), a partially structured intermediate state (I) and a globular native state (N). The unfolded and partially folded proteins can form aggregate species that include highly disordered aggregates, as well as amyloid fibrils that form through a nu-cleation and growth mechanism. Adapted from (Hartl and Hayer-Hartl, 2002). 4.4 The heat-shock response The heat-shock response is a gene regulatory program that counteracts the build-up of misfolded proteins by upregulating the expression of factors that promote protein folding. This transcriptional program is driven by a conserved transcription factor called Heat shock factor 1 (Hsf1) (Anckar and Sistonen, 2011; Mager and De Kruijff, 1995). Exposure to stressful conditions, such as heat shock, causes cells to accumulate misfolded proteins that in turn trigger Hsf1 activation. Active Hsf1 binds the promoters of the heat-shock response genes and induces their transcription. The folding promoting factors that are induced decrease the concentrations of stressful misfolded proteins and restore homeostasis. Thus, the heat-shock response is transiently induced in response

15

to exposure to conditions of protein folding stress. Yeast expresses a single essential Hsf1 homologue, while other organisms may express more (Miller and Mittler, 2006; Verghese et al., 2012). Hsf1 consists of a DNA-binding domain, three leucine zipper repeats that are responsible for trimerization, and a carboxyl-terminal transactivation domain. Hsf1-responsive promoters are identified by the presence of an upstream activating sequence (5'-nGAAn-3') known as the ‘‘heat shock response element’’ (HSRE) (Sorger and Pelham, 1987). These HSREs are organized as either continuous or discontinuous units of nGAAn repeats. In unstressed cells, Hsf1 is localised in both the cytosol and the nucleus and is required for both house-keeping and stress-induced transcriptional activation. In response to applied stress, Hsf1 assembles into a trimer, becomes hyperphosphorylated, accumulates within the nucleus, and binds to HSREs. The result is an increase of the rate of transcription by a factor of 100 (Mager and De Kruijff, 1995). Hsf1 target genes are mainly involved in promoting protein folding and protein degradation (Hahn et al., 2004; Yamamoto et al., 2005). Many of the proteins expressed from genes with HSRE-containing promoters were intially identified as polypeptides that are upregulated following a heat shock, and these proteins have consequently been named ‘‘heat-shock proteins’’ (HSPs) (Morimoto, 1993; Sorger and Pelham, 1988; Verghese et al., 2012). However, it is important to note that other transcription factors than Hsf1, such as the yeast transcription factor Msn2/4, may also trigger the induction of genes in response to heat stress (Martinez-Pastor et al., 1996; Schmitt and McEntee, 1996). 4.5 Proteostasis Protein homeostasis, which is often abbreviated as ‘‘proteostasis’’, is the cellular process of controlling the conformations, concentrations, interactions and locations of proteins, through mainly transcriptional and translational changes (Balch et al., 2008). The proteostasis network consists of several integrated and competing biological pathways that are associated with protein synthesis, folding, trafficking and degradation (Labbadia and Morimoto, 2015). Cells are exposed to a wide range of environmental and metabolic conditions that trigger proteostasis imbalance by inducing protein misfolding and aggregation. Under such imbalance, the proteostasis network responds by restoring proteostasis (Balch et al., 2008). A prime example of such regulation of the proteostais

16

network is the above described heat-shock response. In brief, cells maintain proteostasis by regulating the balance between the production of misfolded proteins and their clearance by folding and degradation. An accumulation of toxic proteins impairs proteostasis and may lead to metabolic, oncological, neurodegenerative and cardiovascular disorders (Balch et al., 2008). Genetic diseases linked to protein misfolding are caused, in principle, by either the loss or gain of function. Gain of function is reflected by the gain of proteotoxicity in many neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), Alzheimer’s disease (AD) and polyglutamine (PolyQ) expansion. Loss of function diseases include cystic fibrosis and Gaucher disease, and are caused by malfunction of the proteostasis network due to mutations that impair protein folding and degradation (Cohen and Kelly, 2003). 4.6 Molecular chaperones The term ‘‘molecular chaperone’’ was first used to describe a protein that prevents incorrect interactions between the histones and DNA in amphibian eggs (Laskey et al., 1978). Extension of the concept of molecular chaperones came from the study of the Rubisco assembly, which revealed a protein that binds Rubisco and prevents incorrect protein interactions (Musgrove and Ellis, 1986). Today, the most commonly used definition of a molecular chaperone is a protein that binds to client proteins and promotes their folding to their native states (Hartl et al., 2011; Hartl and Hayer-Hartl, 2002). These molecular chaperones arose early during evolution: they suppress protein aggregation during folding and maintain proteins soluble (Hartl et al., 2011; Kim et al., 2013). Moreover, when mutations destabilise the protein structure, chaperones act as a buffer and therefore facilitate the evolution of new functions and phenotypic behaviour (Tokuriki and Tawfik, 2009). Molecular chaperones constitute a group of functionally but not evolutionary related proteins. According to their molecular weight, they are divided into several classes. Proteins in same class often show high sequence homology and are structurally and functionally related. In contrast, there is little sequence homology between chaperones of different classes (Walter and Buchner, 2002). Molecular chaperones have diverse cellular functions including facilitating de novo protein folding, refolding of misfolded proteins, oligomer assembly,

17

intracellular transport and protein degradation (Hipp et al., 2014; Vabulas et al., 2010; Verghese et al., 2012). These common functions of molecular chaperones are shown in Figure 3 and Table 1. Generally, chaperones bind proteins that expose hydrophobic segments on their surface and thereby suppress aggregation and promote folding by inducing conformational changes. A frequently occurring theme is that the substrate binding by the chaperone is regulated by adenosine-nucleotide binding and hydrolysis, with different substrate affinities between the ATP and the ADP binding conformations (Walter and Buchner, 2002). Thus, many chaperones require ATP for their function.

Figure 3: The function of chaperones. Proteostasis is maintained by balancing the functional protein pool in the cell. Chaperones are involved in protein folding, degradation and disaggregation. Pathway in green are pathways of biogenesis, those in red are degradation pathways, and those in purple are conformational maintenance (Hipp et al., 2014). Many molecular chaperone family members are induced by heat shock and are consequently named Hsps. Hsp family members include abundant constitutively expressed housekeeping isoforms, as well as stress-inducible

18

isoforms. Hsps include several structurally distinct classes including Hsp40, Hsp60, Hsp70, Hsp90, Hsp100 and small Hsps (Becker and Craig, 1994). The major Hsps in yeast are summarised with their associated functions in Table 1. They all function in the proteostasis network to protect cells from misfolded proteins (Becker and Craig, 1994; Schlesinger, 1990). After acute proteostasis imbalance, the induced Hsps reactivate the structurally damaged proteins and facilitate the degradation of folding resilient species. Table 1: Major molecular chaperones in the yeast cytosol

Chaperone family

Protein

ATPase

Function

Hsp70

Ssa1/2/3/4 Ssb1/2

Yes

Protein folding, protein degradation, de novo protein

folding1

Hsp110 Sse1/2 Yes NEF for Hsp70 and holdase

activity2

Hsp40

Ydj1, Sis1

No Co-chaperone for Hsp70 ATPase activity, binds misfolded proteins3

Hsp90 Hsc82 Hsp82

Yes

Binds misfolded proteins, promotes refolding4 and

degradation5, client protein maturation6 and proteasomal

intergrity7 Hsp100 (AAA+ ATPase)

Hsp104

Yes

Disaggregation8

Small Hsps

Hsp26, Hsp42

No

Renders aggregates more accessible to Hsp104/Hsp70/Hsp409, promotes resolubilisation10, sequestration of misfolded

proteins11 1 Hartl. F.U. Molecular chaperones in cellular protein folding. Nature 381, 571-579 (1996). 2 Shaner et al. The yeast Hsp110 Sse1 functionally interacts with the Hsp70 chaperones Ssa and Ss. J Biol Chem 280, 41262-41269 (2005). 3 Cyr, D. M. et al. DnaJ-like proteins: molecular chaperones and specific regulators of Hsp70. Trends in Biochem Sci 19. 176-181 (1994). 4 Nathan, D.F et al. In vivo functions of the Saccharomyces cerevisiae Hsp90 chaperone. Proc Natl Acad Sci U S A 94. 12949-12956 (1997). 5 McClellan, A. J et al. Folding and quality control of the VHL tumour suppressor proceed through distinct chaperone pathways. Cell 121, 739-748 (2005). 6 Nathan, D.F & Lindquist, S. Mutational analysis of Hsp90 function: interactions with steroid receptor and a protein kinase. Mol Cell Biol 15(7): 3917-25 (1995). 7 Imai, J et al. 2003 The molecular chaperone Hsp90 plays a role in the assembly and maintenance of the 26S proteasome. EMBO J 15;22(14): 3557-67 (2003). 8 Glover, J. R & Lindquist, S. Hsp104, Hsp70 and Hsp40: a novel chaperone system that rescues previously aggregated proteins. Cell 94, 73-82 (1998). 9 Cashikar, A. G et al. A chaperone pathway in protein disaggregation. Hsp26 alters the nature of protein aggregates to facilitate reactivation by Hsp104. J Biol Chem 280. 23869-23875 (2005). 10 Haslbeck, M et al. Some like it hot: the structure and function of small heat-shock proteins. Nat Struct Biol 12, 842-846 (2005). 11 Specht, S et al. Hsp42 is required for sequestration of protein aggregates into deposition sites in Saccharomyces cerevisiae. J Cell Biol 195, 617-629 (2011).

19

4.7 Hsp70 molecular chaperones Hsp70 is an essential class of heat shock proteins that assists in protein folding and degradation. Hsp70 is involved in a wide range of functions including the de novo folding and refolding of proteins, as well as in the assembly and disassembly of oligomeric proteins and translocation over cellular membranes (Hartl and Hayer-Hartl, 2002; Mayer and Bukau, 2005). Hsp70 facilitates the folding of protein substrates by repeated interactions via its substrate-binding domain (SBD) (Hartl and Hayer-Hartl, 2002; Mayer and Bukau, 2005). Nucleotide binding in the nucleotide-binding domain (NBD) allosterically regulates substrate interactions with the SBD. When ATP is bound, the SBD adopts an open conformation that has low affinity for substrates and high on and off rates. In contrast, when ADP is bound, the SBD attains a closed conformation that has a higher affinity for substrates with reduced on and off rates. Figure 4 illustrates the complete Hsp70 folding cycle. Upon interaction with protein substrate, hydrolysis of ATP is stimulated and the substrate is trapped. Exchange of ADP for ATP opens the SBD and releases the substrate. The basic Hsp70 ATPase cycle is regulated by cofactors that interact with the NBD and that are essential for the activity of the chaperone (Mayer and Bukau, 2005). Cofactors of the Hsp40 class accelerate nucleotide hydrolysis, while nucleotide exchange factors (NEFs) accelerate nucleotide exchange. Both types of cofactors are present in yeast and their functions are presented in more detail below.

Figure 4: The Hsp70 cycle of protein folding with two conformational states. Adapted from (Mayer and Bukau, 2005).

20

4.8 Hsp70 in yeast The Hsp70 family in yeast includes eleven members (Table 2). Hsp70 is involved in many functions, notably the de novo folding of proteins, assembly and disassembly of protein complexes, transport of proteins across biological membranes, protein refolding and degradation of misfolded proteins (Boorstein et al., 1994; Kabani and Martineau, 2008; Mayer and Bukau, 2005). The chaperone is present in several cellular compartments including the cytosol, the endoplasmic reticulum and mitochondria. The endoplasmic reticulum expresses only one Hsp70, Kar2, (Normington et al., 1989), while mitochondria harbour three distinct Hsp70s: Ssc1, Ssc3 and Ssq1, which have distinct and rarely overlapping functions (Verghese et al., 2012). The yeast cytosol expresses seven Hsp70s, based on sequence homology, belong to the three subfamilies: stress seventy subfamily A (Ssa1, Ssa2, Ssa3 and Ssa4), stress seventy subfamily B (Ssb1 and Ssb2) and stress seventy subfamily Z (Ssz1) (Werner-Washburne and Craig, 1989). The Ssa family members are highly homologues and inactivation of all four genes results in non-viable cells. Ssa2 is constitutively expressed, while Ssa1, Ssa3 and Ssa4 are stress inducible. The stress-induced forms are transcriptionally regulated by Hsf1 (Sorger, 1991; Sorger and Pelham, 1988). Ssa2 is expressed at twice the level of Ssa1, while Ssa3 and 4 are not expressed under non-stressful conditions. However, in genetic experiments the expression of either Ssa3 or Ssa4 can compensate for the functional loss of Ssa1 and Ssa2 (Craig and Jacobsen, 1984; Kabani and Martineau, 2008; Werner-Washburne and Craig, 1989). Thus, the Ssa family members exhibit at least partially redundant functions. Ssb1 and Ssb2 are constitutively expressed Hsp70s that associate with nascent polypeptides during protein synthesis and support co-translational folding (Willmund et al., 2013). Functional interactions at the ribosome exit tunnel are facilitated by the ribosome-associated complex (RAC), a stable, ribosome-associated heterodimer of Ssz1 and Zuo1 (Gautschi et al., 2001; Koplin et al., 2010; Wegrzyn and Deuerling, 2005). Ssz1 exhibits homology to the NBD of Hsp70, and Zuo1 is an Hsp40 that, together with Ssb1 or Ssb2, forms a ribosome chaperone triad (Gautschi et al., 2002). Ssb1 and Ssb2 differ by only four amino acids and exhibit 60% amino acid identity with the Ssa subfamily (Boorstein et al., 1994). Cells lacking either Ssb1 or Ssb2 have no phenotypes,

21

but the double null is slow growing at lower temperatures, suggesting that both chaperones have redundant functions (Craig and Jacobsen, 1985). The function of Ssb1 and Ssb2 appears to be to hold the nascent polypeptide chains that emerge from the ribosome and protect them until they meet downstream chaperones. Table 2: The Hsp70 family of chaperones in S. c e r ev i s iae . Hsp70 exists in several compartments of eukaryotic cells, mainly in the cytosol, mitochondria and endoplasmic reticulum. Details of Hsp70 are given below.

Protein Localisation Transcription Note Ssa1 Cytosol Stress inducible Ssa2 Cytosol Constitutively expressed Ssa3 Cytosol Stress inducible Ssa4 Cytosol Stress inducible Ssb1 Cytosol Constitutively expressed Ssb2 Cytosol Constitutively expressed

Ssz1 Cytosol Constitutively expressed Not a bona fide

Hsp70 chaperone

Kar2 Endoplasmic

reticulum Constitutively expressed

Ssc1 Mitochondria Constitutively expressed Ssc3 Mitochondria Constitutively expressed Ssq1 Mitochondria Constitutively expressed

4.9 Co-chaperones of Hsp70 4.9.1 Hsp40 Hsp40s (otherwise known as ‘‘J-domain proteins’’) deliver substrates to Hsp70 and simultaneously accelerate ATP hydrolysis. Their activities are essential for the chaperone activities of Hsp70 (Mayer and Bukau, 2005; Szabo et al., 1996). All Hsp40s carry a highly conserved J-domain of length 75 amino acids, named after the archetypical bacterial Hsp40 DnaJ (Kampinga and Craig, 2010). Biochemical experiments have shown that the J-domain is sufficient to stimulate the ATPase activity of Hsp70 (Wall et al., 1994). The J-domain interacts with the Hsp70 NBD and induces conformational changes to close

22

the Hsp70 substrate-binding domain (Kampinga and Craig, 2010). Hsp40 binds substrates, but generally with lower affinity than Hsp70. Further, when the co- chaperone is in close proximity of Hsp70, protein clients are transferred from Hsp40 to the Hsp70. Hsp40s regulate the formation of Hsp70 and substrate complexes in three ways (Cheetham and Caplan, 1998; Cyr et al., 1994; Fan et al., 2003). Firstly, Hsp40s have a peptide-binding domain that interacts with non-native proteins and delivers them to Hsp70 by binding to the chaperone. Secondly, Hsp40s stabilize the substrate-Hsp70 complex by accelerating the ATPase activity of the Hsp70. Finally, Hsp40s function at different sites within the cell (Fan et al., 2003). Thus distinct Hsp40s interact with Hsp70 and enable the binding of unique client proteins. In the yeast genome, 22 Hsp40 genes have been identified and are classified into types I, II and III (Li et al., 2009). They are classified mainly based on the position of the J domain in the protein. The J domain is placed at the N-terminus in all types. Type I Hsp40s possess the J-domain, a glycine and phenylalanine rich (G/F-rich) region, a cysteine rich region and a zinc finger-like domain, followed by a C-terminal domain (CTD). Type II Hsp40s contain a J-domain and a G/F-rich region followed by CTDs I and II. Type III Hsp40s carry only the J-domain as indicated in Figure 5 (Fan et al., 2003; Summers et al., 2009). Substrate specificity among the different classes in vivo is not well understood. Two Hsp40s, Ydj1 (type I) and Sis1 (type II), play a prominent roles in the yeast cytosol. Recent studies have shown that Ydj1 is involved in the suppression of protein aggregation, while Hsp70-Sis1 promotes protein degradation (Summers et al., 2013). Studies on Sis1 have shown that its interaction with Hsp70 is a strict requirement for the ubiquitylation of misfolded proteins (Shiber et al., 2013). Similarly, sufficient cellular levels of Sis1 are required for the delivery of misfolded proteins to the nucleus for proteasomal degradation (Park et al., 2013). In vitro studies on metazoan Hsp40s combined with Hsp70-Hsp110 have revealed that they form a large complex that functions as a powerful disaggregation machinery (Nillegoda et al., 2015; Rampelt et al., 2012). These experiments suggest that Hsp40s regulate the Hsp70 disaggregation capacity.

23

The relevance of these findings for the yeast system is unclear. Curiously, some proteins other than Hsp40s carry a J-domain. The cyclin Cln3 carries a functional J-domain required for cell cycle regulated interactions that promote the degradation of the cyclin by the ubiquitin-proteasome system (Truman et al., 2012; Yaglom et al., 1996).

Figure 5: Domain organisation for Hsp40 subtypes. J represent, J-domain; G/F, glycine and phenylalanine-rich region; ZFLR, Zinc finger-like region; CTD1, carboxyl-terminal domain I; CTDII, carboxyl-terminal domain II; DD, dimerization domain. Adapted from (Fan et al., 2003). 4.10 Nucleotide-exchange factors

NEFs accelerate the exchange of ADP to ATP in Hsp70, resulting in the concomitant opening of the substrate-binding domain (Kim et al., 2013). Eukaryotic NEFs exhibit highly diverse structures and they do not share any common defining domain between the NEF families (Figure 6) (Bracher and Verghese, 2015). Despite their structural heterogeneity, NEFs function by locking two lobes of the Hsp70 NBD in a distorted conformation, resulting in decreased affinity for ADP. Binding of ATP completes the exchange reaction and releases the NEF from Hsp70. The nucleotide exchange drives allosteric interdomain conformational changes in Hsp70, so that the SBD releases the bound substrate (Hartl et al., 2011). Sse1, Sse2, Snl1 and Fes1 are members of three structurally unrelated families of NEFs present in the yeast cytosol (Verghese et al., 2012). Each of the NEFs has been shown to interact with Hsp70 in vivo and in vitro (Dragovic et al., 2006a; Dragovic et al., 2006b; Kabani et al., 2002a; Shaner et al., 2005; Sondermann et al., 2002b). The presence of several NEFs from three families

J G/F Z F L R CTD2

J G/F CTD2G/M CTD1 DD

J

I

II

III

24

suggests that they have specialized in the cell. Table 3 summarizes the NEFs in yeast cells and their phenotypes. Each NEF family is presented under separate headings below.

Figure 6: Domain organisation of Hsp70 and its NEFs. Where, NBD, Nucleotide binding domain; SBD, Substrate binding domain; TMD, Transmembrane binding domain; N, Amino terminal, C, Carboxyly terminal. Indicating the difference in domain organisation with each NEFs in yeast. Adapted from (Bracher and Verghese, 2015; Xu et al., 2012). Table 3: Hsp70 NEFs in yeast and their phenotypes

4.10.1 The Hsp110 family – Sse1 and Sse2 Hsp110s are highly abundant NEFs present in the yeast cytosol, and belong to the Hsp70 superfamily. The two Hsp110s in yeast, Sse1 and Sse2, are 97% similar and have 70% homology to Hsp70 (Mukai et al., 1993a). Sse1 is expressed at 71,700 molecules per cell in logarithmically growing cells and is the most abundant NEF, while Sse2 is expressed at 6,300 molecules per cell (Ghaemmaghami et al., 2003). Sse1 makes stable complexes with both the Ssa

Armadillo repeats Fes1

Hsp70

Hsp110

NBD

NBD

SBDβ SBDα

SBDβ SBDα

Snl1TMD Bag-1 domain

Linker

N C

Family Yeast protein Phenotype

Hsp110 Sse1 Generally slow-growing (Shirayama

et al., 1993) Synthetically lethal with sse2

Sse2 No phenotype (Mukai et al., 1993b) Synthetically lethal with sse1 (Shaner

et al., 2004)

BAG Snl1 No phenotype (Ho et al., 1998)

HspBP1 Fes1 Slow-growing at elevated

temperatures (Kabani et al., 2002a)

25

and Ssb class of Hsp70 (Raviol et al., 2006). Yeast cells lacking Sse1 have severe growth defects, whereas Sse2 deletion has no effect. Deletion of both genes is lethal (Mukai et al., 1993b; Shaner et al., 2004). Thus, the high expression levels and severity of the phenotypes suggest that the Hsp110 family is responsible for most NEF activity in the yeast cytosol. Even though Hsp110s are structural homologues of Hsp70 and therefore a potential molecular chaperone, their only firmly established function is to provide NEF activity to canonical Hsp70s (Dragovic et al., 2006a; Raviol et al., 2006). Hsp110 family proteins consist of an NBD, a β-sandwich domain and a

triple α−helical bundle domain (Liu and Hendrickson, 2007). The crystal structure (Figure 7) shows that Sse1 is in complex with the Hsp70 NBD during nucleotide exchange (Polier et al., 2008; Schuermann et al., 2008). Sse1 in the ATP-bound state embraces the NBD of Hsp70 by face-to-face interactions between the two NBDs and interactions between the side of the Hsp70 NBD and the Sse1 triple α-helix bundle domain (Andréasson et al., 2008). The result is an Hsp70 NBD with twisted NBD lobes, which has low affinity for ADP. Looking at the conformations and structural dynamics of the Hsp70 NBD, complexes with Sse1 and BAG NEFs induce very similar changes (Andréasson et al., 2008; Polier et al., 2008; Schuermann et al., 2008). Thus, both Hsp110 and BAG employ the intrinsic structural repertoire of the Hsp70 NBD to lock the domain in a conformation with low affinity for nucleotide. Functions of Hsp110 in addition to the well-characterized NEF activity have been proposed, but remain controversial. Mammalian Hsp110 binds and protects other proteins from heat-denaturation and thus displays holdase activity (Easton et al., 2000). Sse1 displays holdase activity also in yeast (Goeckeler et al., 2002). Attempts to characterize the holdase activity of Sse1 have been inconclusive. Sse1-substrate interactions appear to be activated by heat, suggesting that the unfolded protein interacts specifically with other proteins in vitro (Polier et al., 2010). Peptide-binding studies suggest that aromatic peptides interact with Sse1 (Xu et al., 2012). Nevertheless, the addition of Sse1 to Hsp70-Hsp40 refolding reactions potentiates the chaperone system to reactivate aggregated proteins (Goeckeler et al., 2002; Rampelt et al., 2012; Shorter, 2011).

26

Figure 7: Crystal structure of yeast Sse1. The Sse1 is in green complex with Hsp70 NBD in dark blue (PDBID -3D2F) (Polier et al., 2008). 4.10.2 The BAG family – Snl1 Snl1 is the only Hsp70 NEF in yeast that has a BAG (Bcl2-associated athanogene) domain. The domain was first described in BAG-1, a mammalian protein originally described to be associated with the anti-cell death protein Bcl2 (Takayama et al., 1995). It was subsequently found that the evolutionarily conserved BAG domain interacts with the NBD of Hsp70 and functions as an NEF (Sondermann et al., 2002b). The structure of the complex consisting of the triple α-helical BAG domain and the NBD of Hsp70 has been determined by crystallography (Figure 8) (Sondermann et al., 2001). The BAG domain locks the NBD in an open conformation reminiscent of the structure induced by Hsp110. The similarity in the NBD conformations induced by Snl1 BAG and Hsp110 Sse1 is supported by structural dynamics studies using hydrogen-deuterium exchange (Andréasson et al., 2008). From biochemical studies, Snl1 was found to interact with both the Ssa and Ssb classes of Hsp70 and to trigger nucleotide exchange (Sondermann et al., 2002a). In yeast, Snl1 localises predominantly to the nuclear envelope and endoplasmic reticulum, and is anchored to the membrane through an amino terminal single-spanning transmembrane domain (Ho et al., 1998). The cellular function of Snl1 is still unknown and snl1Δ cells exhibit no apparent phenotype. Overexpression of Snl1 suppresses temperature-sensitive mutations in nucleopore components, and the activity is linked to its ability to function as an NEF (Sondermann et al., 2002b). Effects of overexpression of the BAG

27

domain in isolation are also seen on prion propagation (Kumar et al., 2014). However, the physiological relevance of the overexpression phenotypes is not known. Curiously, the soluble BAG domain of Snl1 has been found to associate with intact ribosomes (Verghese and Morano, 2012). Again the biological significance of this finding is not clear. In animal and plant cells, proteins that carry BAG domains are involved in apoptosis, DNA binding and transcription (Alberti et al., 2003; Kabbage and Dickman, 2008).

Figure 8: Crystal structure of the Bag domain. The Bag domain is shown in green and the three α−helix complex with Hsc70 in dark blue (PDBID- 1HX1) (Sondermann et al., 2001). 4.10.3 The HspBP1 family – Fes1

Fes1 belongs to the HspBP1 (Hsp70-binding protein 1) family of NEFs. This family contains, together with Hsp110, the most highly conserved eukaryotic NEFs (Shomura et al., 2005). They are structurally characterized by a central armadillo repeat domain that interacts with Hsp70. A crystal structure of the central core of the HspBP1 armadillo domain in complex with the NBD of Hsp70 shows that the concave surface of the NEF binds lobe II of the Hsp70 NBD to reduce its affinity for nucleotide (Figure 9) (Shomura et al., 2005). Hydrogen-exchange studies show that part of lobe II and the entire lobe I become completely destabilized by interactions with Fes1 (Andréasson et al., 2008). This mechanism of nucleotide exchange is unique, since it relies on binding to lobe II and the consequent destabilization of lobe I.

28

Figure 9: Crystal structure of HspBP1. The core domain of HspBP1 in green is in complexed with the fragment of Hsp70 ATPase domain indicated in blue (PDB ID – 1XQS) (Shomura et al., 2005). In yeast, Fes1 was initially identified as the cytosolic homologue of the endoplasmic reticulum NEF Sil1. However, Fes1 is localized to the cytoplasm and binds cytosolic Hsp70 (Dragovic et al., 2006b; Kabani et al., 2002a). Fes1 is 38% similar and 25% identical to human HspBP1. Even though both Fes1 and its mammalian homologue HspBP1 act as NEFs for Hsp70, they appear to have distinct cellular functions (Kabani et al., 2002b). From in vitro chaperone mediated refolding assay it is evident that HspBP1 inhibits refolding whereas, Fes1 is not involved (Kabani et al., 2002b). HspBP1 also inhibits CHIP ubiquitin ligases, however cellular roles of Fes1 are elusive (Alberti et al., 2004). Hence, both Fes1 and HspBP1 have different functional roles and the exact mechanism remains unclear. The cellular function of Fes1 has been unclear. Cells lacking Fes1 exhibit defects in growth at higher temperatures and induce the heat-shock response (Abrams et al., 2014; Kabani et al., 2002a). The growth phenotype of the null variant is phenocopied by two mutations (A79R and R179A) that abolish the NEF interaction with Hsp70, indicating that the phenotypes are related to exchange activity (Shomura et al., 2005). Early studies proposed that Fes1 functions at the ribosome, and in vitro interaction studies revealed interactions with the Ssa class and with the ribosomally associated Ssb class (Dragovic et al., 2006b). In contrast, later studies employing copurifications from yeast lysates did not detect an interaction with the Ssb class or ribosomes (Verghese and Morano, 2012). Thus, the function of Fes1 at the ribosome and in de novo folding is unclear. Similarly, Fes1 is not required for Hsp70-dependent and Hsp104-mediated disaggregation and reactivation of proteins following heat

29

shock (Abrams et al., 2014). A parallel line of functional studies involves prion biology. Notably, Fes1 is required for prion formation and curing (Kryndushkin and Wickner, 2007). In contrast, overexpression of Fes1 causes prions to propagate better by inducing Hsp70 to its open conformation (Jones et al., 2004). It is probable that the effects of Fes1 on prion biology stem from its effects on the Hsp70 system as an NEF.

4.11 The ubiquitin-proteasome system One way to maintain proteostasis is by directing toxic misfolded proteins to degradation through the ubiquitin-proteasome system (UPS) (Wolf and Hilt, 2004). Here, proteins in the cytosol and nucleus are subjected to degradation via polyubiquitylation, which serves as a signal for degradation by the proteolytic 26S proteasome. Hence, reduced levels of misfolded proteins in the cell help to control proteostasis. The ubiquitylation process relies on the sequential action of the E1 (ubiquitin-activating enzymes), E2 (ubiquitin-conjugating) and E3 (ubiquitin ligases) classes of enzymes. The C-terminus of ubiquitin is activated by the ubiquitin-activating enzyme E1 (Uba1) with the consumption of ATP, to form an E1-thiol ester intermediate. Activated ubiquitin is transferred to the ubiquitin-conjugating enzyme (E2) (Ubc enzyme) by transesterification of a cysteine residue. Subsequently, ubiquitin is transfered from E2 to E3, to be subsequently delivered to the protein substrates by the formation of an isopeptide bond between the C-terminus of ubiquitin and a lysine residue on the protein. The polyubiquitin chain assembly often occurs between seven lysines of ubiquitin, which act as acceptors of isopeptide bonds (K6, K11, K27, K29, K33, K48, K63). In general, lysine linkage is an indicator of differential signalling, whereas K48 chain extension is devoted to targeting proteins for degradation. This is essential for cell viability. K63 chains are dispensable under non-stressful conditions and do not affect the proteasomal degradation of proteins, but are required for DNA-damage response (Peng et al., 2003; Xu et al., 2009). Extended chains of ubiquitin with atleast four-ubiquitin polyubiquitin chains are subject to proteasomal degradation (Figure 10) (Hershko, 1996; Hochstrasser, 1995; Ravid and Hochstrasser, 2008; Wolf and Hilt, 2004). The ubiquitin enzymes E1, E2 and E3 act in a sequential manner (Figutre 10). There is only one E1 enzyme in yeast, with eleven E2s and 60-100 E3s (Finley

30

et al., 2012). The UBA1 gene codes for the ubiquitin-activating enzyme (E1) in yeast, while a family of UBC genes encodes the ubiquitin-conjugating enzyme (E2). The ubiquitin ligase (E3) family is the largest ubiquitin family, and its members add selectivity to the process of degradation. Together, they form a complex and hierarchical system that directs the misfolded proteins to ubiquitin-dependent degradation.

Figure 10: Schematic representation of the UPS. Sequential action of ubiquitin E1, E2 and E3 enzymes on substrate for polyubiquitinylation and directs to the proteasome. Adapted from (Hershko, 1996; Varshavsky, 1997). Ubiquitin ligases of relevance to the work presented here are Ubr1 and San1. These enzymes are involved in the cytoplasmic and nuclear degradation of misfolded proteins (Eisele and Wolf, 2008; Gardner et al., 2005; Heck et al., 2010; Prasad et al., 2010). Both Ubr1 and San1 belong to RING-domain ubiquitin ligases, a family with 44 members in yeast. Substrate recruitment is executed either by direct binding by specialized domains of the ubiquitin ligase or via adapter proteins (Deshaies and Joazeiro, 2009). Ubr1 localises to the cytosol and recognizes substrates via binding of their different N-terminal amino acid residues (Bartel et al., 1990; Kim et al., 2014). Specifically, unacetylated N-terminal methionines followed by a hydrophobic residue function as a recognition site. Since the N-terminal residue determines the half-lifes of proteins in a Ubr1-dependent way, the degradation system is named the “N-end rule pathway”. Functioning in parallel to the cytosolic Ubr1, San1 has emerged as one of the main nuclear ubiquitin ligases that targets misfolded proteins for degradation (Gardner et al., 2005; Heck et al., 2010; Rosenbaum et al., 2011)). San1 directly recognizes its misfolded substrates by interactions via its intrinsically disordered N- and C-terminal domains with hydrophobic patches. Thus, both Ubr1 and San1 target proteins for degradations via direct interactions with hydrophobic residues.

31

4.12 Deubiquitylation Deubiquitinating enzymes are specialised proteases that reverse the attachment of ubiquitin to proteins. These enzymes catalyse the hydrolysis of isopeptide bonds between ubiquitin and proteins (Reyes-Turcu et al., 2009). There are 20 deubiquitinating enzymes (DUBs) in yeast (Finley et al., 2012). These DUBs regulate the ubiqiuitin-dependent processes by cleaving ubiquitin-protein bonds. The main function of DUBs is to recycle ubiquitin from polyubiquitin chains that serve as signals for proteasomal degradation. Defects in this process give rise to reduced ubiquitin levels and perturb proteostasis. In yeast, Upb3, Ubp6, Rpn11 and Doa4 serve as the main DUBs that release ubiquitin before proteins are targeted for proteasomal degradation (Hanna et al., 2006; Swaminathan et al., 1999). Ubp3 is a one of the main deubiquitination enzymes, and Ubp3-deficient cells accumulate ubiquitin conjugates (Baxter and Craig, 1998). UBP3 was initially identified as a high-copy suppressor of the temperature-sensitive phenotype of yeast cells that lack the major cytosolic Hsp70 chaperones Ssa1 and Ssa2 (Baxter and Craig, 1998). Overexpression of Ubp3 results in impairment of degradation of certain misfolded proteins, and therefore gives them further opportunities to fold (Oling et al., 2014). Hence, deubiquitylation also plays a role in maintaining proteostasis. 4.13 The 26S proteasome The proteasome is an important component of the proteolytic machinery in eukaryotic cells. The proteasome preferentially degrades proteins that are covalently tagged with polyubiquitin chains (Hershko et al., 1982). The degradation process involves several linked ATP-hydrolysis-dependent activities, including polyubiquitin chain binding, deubiquitylation and protein unfolding. The proteasome has a high affinity for ubiquitin chains that consist of four or more moieties (Thrower et al., 2000). After translocation into the central proteolytic chamber of the proteasome, proteolysis results in peptide products of length 3-20 amino acids, and these are further processed by endo- and amino-peptidases to give single amino acids (Tamura et al., 1998). The 26S proteasome (2.4 MDa) is made up of a 20S catalytic core particle and a pair of 19S regulatory particles that are located on both ends of 20S proteasome

32

(Pickart and Cohen, 2004). The catalytic core particle consists of two central β-

rings that display catalytic activity, and two distal α-rings that control the passage of substrate for degradation. The proteolytic active sites are buried inside the core particle to ensure specific interactions, and to limit non-specific proteolysis (Groll et al., 2005). The 19S regulatory particles recognise substrates and interact with shuttle factors that deliver substrates to the proteasome. Shuttle factors have one or more distinctive UBA domains that bind polyubiquitin chains, and an N-terminal UBL domain that interacts with the proteasome. The proteasome is required for the function of many cellular processes and provides the major proteolytic activity in the cytosol and nucleus (Demartino and Gillette, 2007). A key function is to maintain proteostasis by degrading misfolded or damaged proteins. The subcellular distribution of the 26S proteasome appears to be different in yeast and mammalian cells, perhaps because of differences regarding closed and open mitosis (Russell et al., 1999). In logarithmically growing yeast cells, proteasomes are primarily localised to the nucleus (Huh et al., 2003) and are targeted to the nucleus by means of signals present in several α core subunits (Tanaka et al., 1990). Thus, the nucleus carries the bulk of proteolytic activity in yeast cells. 4.14 Protein quality control in the cytosol and nucleus Protein quality control (PQC) is the process in which proteostasis is maintained by the selective removal of misfolded proteins from the cell. Such damaged proteins are either repaired by chaperone-dependent refolding or proteolytically eliminated by PQC networks (Buchberger et al., 2010). During protein biosynthesis, PQC plays an important role, removing aggregation-prone and potentially toxic polypeptides that are resilient to folding into their native structures. Preferentially, misfolded proteins are subjected to refolding attempts by chaperones, and if the protein fails to attain its native conformation it is targeted for degradation by the ubiquitin-proteasome system (UPS) (Doyle et al., 2013; Gottesman et al., 1997). A proteolytic system that functions in parallel to the UPS is contained within the lysosome/vacuole. Protein aggregates resistant to UPS degradation are eliminated from the cell via the process of autophagy, which imports the aggregate species into the proteolytically active lysosome/vacuole.

33

In the cytosol, misfolded proteins are recognised by PQC systems primarily because they expose hydrophobic amino acids that are binding sites for chaperones and ubiquitin E3 ligases. Hsp70 plays a major role in PQC, since it is a highly abundant chaperone that ubiquitously interacts with exposed stretches of hydrophobic amino acids (Rudiger et al., 1997). During interaction, Hsp70 shields the hydrophobic stretches from interactions that drive protein aggregation, and thus ensures that the chaperone-associated protein remains soluble. The repeated interactions with Hsp70 and co-chaperones also give the opportunities for substrate to enter productive folding pathways (Kampinga and Craig, 2010). Proteins that do not fold remain transiently associated with chaperones and are targeted for degradation by ubiquitylation or for chaperone-orchestrated aggregation into specialized PQC compartments (Kaganovich et al., 2008; Miller et al., 2015). The mechanisms of triage decisions – whether to refold or to degrade proteins associated with Hsp70 – are poorly understood (Buchberger et al., 2010). Presumably, competing processes of binding other chaperones or ubiquitin E3 ligases result in the process we describe as PQC. Also cofactors of Hsp70 play a role to direct protein substrates to different fates. Studies in yeast using genetically misfolded model proteins have shown that Hsp70 function is required for the ubiquitin-dependent degradation of misfolded proteins (Park et al., 2007; Shiber et al., 2013). Similarly, Hsp40 co-chaperones are also required for the ubiquitin-dependent degradation of misfolded proteins (Kettern et al., 2010; Park et al., 2013; Summers et al., 2013). NEFs have also been implicated in misfolded protein degradation. Cells lacking the major NEF Sse1 fail to degrade misfolded proteins (McClellan et al., 2005a). Thus, the Hsp70 system is central to PQC, but the mechanisms by which it acts and how it is regulated are poorly understood. Misfolded proteins that are not removed from the cell undergo aggregation. During stress-conditions, massive protein misfolding floods the PQC systems and drives aggregation. Interestingly, accumulated aggregates are spatially organized in the cell (Tyedmers et al., 2010). It is possible that such directed and localized aggregation either reduces the toxicity of the proteins or promotes the efficient turn-over of the misfolded proteins (Kaganovich et al., 2008). The formation of protein aggregates is an organized process that is conserved from yeast to mammalian cells. The specific localization of deposits varies between organisms, and depends on their propensity to form aggregates, the surrounding environment, and the stress condition applied (Tyedmers et al., 2010).

34

The aggregates are mainly categorised based on their localisation, and the categories include insoluble protein deposit, (IPOD) and juxtanuclear quality control (JUNQ) entities (Kaganovich et al., 2008; Miller et al., 2015). IPODs are localized adjacent to the vacuole, while JUNQ is localized adjacent to the nucleus. Recent work has shown that JUNQ actually resides within the nucleus and it has consequently been proposed to be renamed as intranuclear quality control (INQ) (Miller et al., 2015). The proteins that constitute JUNQ/INQ are ubiquitylated, mobile and exchange rapidly with the surrounding cytoplasm. In contrast, IPODs are built of terminally aggregated and insoluble proteins that are immobile, such as Htt103Q, and the yeast prions [RNQ] and [URE3] (Kaganovich et al., 2008). Factors that specifically trigger proteins to become incorporated into the different deposits are not well understood, but recent studies have shown that misfolded proteins are targeted to JUNQ/INQ with the help of Btn2 and Hsp42 (Malinovska et al., 2012; Miller et al., 2015). Thus orchestrated aggregation and the transition between soluble and solid phases play key roles in PQC systems. Ubiquitin ligases play a crucial role in targeting misfolded proteins for proteasomal destruction, and these ligases are active in both the cytosol and the nucleus. In yeast, nuclear San1, cytosolic Ubr1/Ubr2/Hul5 and the ribosome-bound ubiquitin ligase Rkr1/Ltn1 perform PQC (Bengtson and Joazeiro, 2010; Eisele and Wolf, 2008; Fang et al., 2011; Nillegoda et al., 2010; Rosenbaum et al., 2011). The degradation pathways that involve these ubiquitin ligases encompass transit of the misfolded proteins between the cytosol and the nucleus. Specifically, Hsp70 and its Hsp40 co-chaperones are required to import misfolded proteins into the nucleus for San1-dependent degradation (Miller et al., 2015; Park et al., 2013; Prasad et al., 2010). Thus, even though San1 resides in the nucleus, many misfolded substrates are cytosolic (Gardner et al., 2005). In addition to classical Hsps, an abundant AAA+ ATPase denoted “Cdc48” has recently been found to play a role in San1-dependent degradation of misfolded proteins to form aggregates (Gallagher et al., 2014). The interface and interactions between the chaperone systems, including Hsp70, and the UPS system are, however, poorly understood. 4.15 Autophagy Autophagy is the process in which cytoplasmic constituents are delivered to the lysosome/vacuole for degradation. Autophagy mainly involves the dynamic

35

rearrangement of cellular membranes to allow a portion of the cytoplasm to be delivered to the lysosome/vacuole (Mizushima and Klionsky, 2007). In yeast, autophagy is strongly induced by nitrogen starvation and (to a lesser extent) by carbon starvation (Takeshige et al., 1992). Autophagy is broadly classified into three categories: microautophagy, macroautophagy and chaperone-mediated autophagy (Cuervo, 2004). Microautophagy is non-selective degradation by the engulfment of small cytosolic components into the lysosome/vacuole; no clear mechanism for this activity has been identified (Kraft et al., 2009). Macroautophagy is the phenomenon in which cytoplasmic components are autophagosized by the formation of a lipid bilayer structure known as the “autophagosome” (Cuervo, 2004). The autophagosome is well characterized in yeast, plant and animal cells. In yeast, 30 ATG genes have been identified that are involved in autophagy (Suzuki and Ohsumi, 2007). Macroautophagy is activated during nutrient starvation (Suzuki and Ohsumi, 2007). Finally, chaperone-mediated autophagy is the process in which selective protein substrates are recognized by Hsp70 (Hsc70) and are taken up by the lysosome through interaction with the LAMP-2A (lysosome-associated membrane protein type 2A) receptor (Cuervo et al., 2004). This process is important in mammalian cells, but has not been described in yeast. Autophagy plays an important role in removing stable protein inclusions that are resilient to the activities of chaperones and the UPS system. 4.16 Alternative splicing in yeast Most genes in higher eukaryotes express pre-mRNA that contains exons and introns, where the exons code for parts of the protein and the introns do not. The biogenesis of functional eukaryotic mRNA requires the removal of intervening introns by RNA splicing. For accurate splicing, the pre-mRNA of eukaryotes from yeast to mammals possesses common motifs that facilitate the recognition, the removal and the joining of exons. Efficient splicing requires a conserved 5' splice site (5'ss, in yeast GTATGT), a branch point (BP, in yeast: TACTAAC), a polypyrimidine tract, and a 3' splice site (3'ss, in yeast TAG) (Ast, 2004). Pre-mRNA splicing occurs by two transesterification reactions, and this conserved mechanism is well understood. The reaction is initiated by forming a phosphodiester bond between the 5'ss and a conserved adenosine residue within the intron at the BP sequence, forming a branched lariat. The second step involves cleavage at the 3'ss, to join the 5' and 3' exons and remove the intron-mature RNA (Figure 11) (Rio, 1993; Ruby and Abelson, 1991). The

36

process of splicing is carried out in a RNA-protein complex known as the “spliceosome” (Rio, 1993). The coding sequences (exons) are joined to form the mature mRNA, which is subsequently exported to the cytoplasm.

Figure 11: Pre-mRNA splicing. Splicing produces mature mRNA by a two-step trans-esterification reaction in the intron region. Exons are shown boxed and the intron as a line. Dashed arrows indicate nucleophilic attack of the hydroxyl group at the splice site. Adapted from (Rio, 1993; Ruby and Abelson, 1991). Alternative splicing extends the gene synthesis capacity to generate isoforms with distinct structures and functions. In alternative splicing, particular exons may be included or excluded from the final, processed mRNA. Examples of functional changes due to alternative splicing include changes in the level of gene expression, intracellular localization and protein stability (Stamm et al., 2005). Evolutionary models for the development of alternative splicing are based on either splicing-hampering mutations that facilitate the skipping of splice sites, or the evolution of novel splicing regulatory factors (Ast, 2004). There are five major forms of alternative splicing: exon skipping, the use of an alternative 5' splice site, the use of an alternative 3' splice site, intron retention, and mutually exclusive exons. These mechanisms are conserved between human and mouse genomes. Exon skipping is the most predominant form of alternative splicing in multicellular organisms, but has not been detected in unicellular organisms (Ast, 2004).

Exon IIExon I GUP PAG(Py)A

Exon I OH Exon IIPAGAPGU

2’OH

+

AGAPGU

OH + PExon I Exon II

5’ Splice site 3’ Splice siteBranch point

5’ splice site cleavage and Lariate formation

3’ splice site cleavage and Exon ligation

37

Splicing is a rare event in yeast and only 4% of genes have introns (Schreiber et al., 2015). Most of the genes with introns have only a single intron (Ares et al., 1999; Parenteau et al., 2008; Spingola et al., 1999). All introns are efficiently removed during gene expression (Ast, 2004). The main function of the yeast introns appears to be to regulate the levels of RNA and protein (Juneau et al., 2006). Alternative splicing is extremely rare in yeast and only a few examples have been described. Alternatively spliced genes include SRC1 (Grund et al., 2008; Rodriguez-Navarro et al., 2002), PTC7 (Juneau et al., 2009), YRA1 (Preker et al., 2002), MER2 (Engebrecht et al., 1991), RPL30 (Vilardell and Warner, 1994) and SUS1 (Cuenca-Bono et al., 2011; Hossain et al., 2011). PTC7 provides the clearest example of alternative splicing, which in this case produces two mRNAs that code for distinct proteins (Juneau et al., 2009). Splicing is regulated by the nature of the carbon source, and the spliced mRNA expresses a protein that localises to mitochondria. The unspliced product, in contrast, localises to the nuclear envelope. The biological significance and function of the isoforms are still unclear. Thus, alternatively spliced genes in yeast are few, which has limited the usefulness of the model organism for the study of the function, mechanism and evolution of alternative splicing. The splicing mechanism is sensitive to the breakdown of proteostasis during heat shock. Previous studies have shown that proteins regulated by heat shock play a role in protecting splicing (Vogel et al., 1995; Yost and Lindquist, 1991). Experiments have shown that Hsp70 mutants overexpress other heat shock proteins, and that Hsp104 in particular facilitates splicing recovery after heat shock. It is important, therefore, to study the roles of Hsp70 and co-chaperones in the protection of splicing during heat stress. 4.17 The model organism

The results presented in this thesis are based on experiments with the model organism Saccharomyces cerevisiae, budding yeast. This is one of the most widely used model organisms for biological studies. Several properties make yeast suitable for biological studies, including its low pathogenicity, rapid growth, ease of mutant isolation, stable haploid and diploid growth forms, well-defined genetic system, and highly efficient DNA transformation system (Gietz and Woods, 2002; Sherman, 2002). S. cerevisiae was the first eukaryotic organism whose genome was completely sequenced (Goffeau et al., 1996). In research, S288c and its derivative strains have been adopted for addressing many differ-

38

ent biological questions (Mortimer and Johnston, 1986). Many human genes related to disease have orthologous versions in yeast, and the organism is well-conserved. Thus, this organism is advantageous to use when studying diverse biological functions (Ploger et al., 2000).

39

5. Aims The overall goal of the work presented in this thesis was to provide insight into the regulation of cytosolic Hsp70 and its role in protein quality control. Specifi-cally, I have focused on characterizing the cellular function of the Hsp70 NEF Fes1. I have addressed the following three aims in individual studies:

1. Define the cellular function of Fes1 in the cytoplasmic Hsp70 system 2. Characterize Fes1 nucleo-cytoplasmic localisation, functional expres-

sion and regulation 3. Provide mechanistic insight into how Fes1 regulates Hsp70 system

In a separate study, I have developed methodology that aims at:

4. Streamlining the assembly of plasmids for recombinant protein expres-sion in bacteria

40

5. 1 Study I 5.1.1 Aim Aim 1: Define the cellular function of Fes1 in the cytoplasmic Hsp70 system This study describes the function of Fes1 as an NEF in Hsp70-dependent PQC. While previous studies have demonstrated the requirement of Hsp70 and its Hsp40 co-chaperones, the role of NEFs in misfolded-protein degradation has been less investigated. Here we show that the NEFs Sse1/2 and Fes1 have specialized roles in the cytosolic PQC system, and Fes1 is essential for misfold-ed protein degradation. The study highlights the role of Fes1 as a factor that determines the fate of protein substrates associated with Hsp70. 5.1.2 Results and discussion Cells lacking Fes1 are hypersensitive and induce the heat-shock response Fes1 is an Hsp70 NEF with a well-characterized biochemical function. Knowledge of the in vivo function of this protein, however, has been limited (Kabani et al., 2002a). In Study I, we found that cells lacking Fes1 cells are hy-persensitive to the presence of conditions that induce accelerated protein mis-folding, including elevated temperatures and the presence of the toxic amino acid analogues azetidine-2-carboxylic acid and canavanine. The hypersensitivity suggests that Fes1 is involved in PQC pathways. Interestingly, we observed that the cells responded to the removal of Fes1 by constitutively upregulating the heat-shock response and accumulating Hsp70,

Hsp90 and Hsp104. Hsf1 was found to be hyperphosphorylated in fes1Δ cells, a hallmark of activation of Hsf1 by proteostasis perturbations. Mutations that

impair the activation of Hsf1 exhibited strong synthetic interactions with fes1Δ, indicating that the induced heat-shock response is required to protect cells lack-ing Fes1. Importantly, the constitutively induced heat-shock response appears to be

unique for fes1Δ cells, and is not induced by other NEF mutations such as

41

sse1Δ, sse2Δ or snl1Δ. Double deletions combining the inactivation of Fes1 and the other NEF’s did not enhance the phenotype, suggesting that the increased sensitivity stems from the loss of a function specific for Fes1 that impairs pro-

teostasis. The unique phenotypes of fes1Δ indicate that Fes1 plays an essential role in maintaining proteostasis and maintaining the heat-shock response repressed. Fes1 interacts with misfolded proteins Direct experimental evidence supporting the suggestion that misfolded proteins

accumulate in fes1Δ cells came from employing the proteostasis and aggregation reporter firefly luciferase fused to GFP (FFL-GFP). We observed that upon a

mild heat-shock, this reporter formed massive aggregates in fes1Δ cells. This observation suggests that Fes1 is important to manage misfolded proteins by suppressing protein aggregation. We screened for interaction partners of Fes1 using a two-hybrid approach and found that Fes1 associated with Hsp70 (Ssa1, Ssa2 and Ssa4, Ssb1), Hsp40 (Sis1), and misfolded protein fragments, including an internal fragment of mis-localized mitochondrial RNA polymerase (Rpo41*; residues T920-L1217). We utilised two Fes1 point mutations (A79R, R1195A) that abolish interaction with Hsp70 to determine whether these substrates interact directly or via Ssa1. Sur-prisingly, we observed that Ssa1 is required for interaction not only with Hsp70 but also with Sis1 and Rpo41*, providing evidence for Fes1-Hsp70-Rpo41* and Fes1-Hsp70-Sis1 ternary complexes. These observations indicate that Fes1 interacts directly with Hsp70 in complex with misfolded proteins. Fes1 is required for the UPS degradation of misfolded proteins We asked whether the degradation of misfolded protein Rpo41* is dependent on Fes1, and answered this by following the protein level after translational arrest with cyclohexamide. Rpo41* was stabilised over a 90-minute time course

in fes1Δ cells. In addition, we asked whether Hsp70 function is required for the degradation, and found that Rpo41* was stabilised also in the ssa1-45 Hsp70 temperature-sensitive strain. Next, we analysed whether the substrate degrada-tion depends on the UPS. We found that Rpo41* is stabilised in strains with

genetically inactivated ubr1Δ and cim3-1, indicating that both an ubiquitin ligase and the proteasome are required for degradation. We proceeded to investigate

42

whether the degradation of other misfolded proteins depends on Fes1. Two mutant versions of dihydrofolate reducatase (DHFR), DHFRmutC and DHFRmutD, which undergo UPS-mediated degradation, were also found to depend on Fes1. The requirement for Fes1 in UPS degradation was specific for misfolded proteins. Several well-folded proteins with previously defined UPS-degradation pathways did not depend on Fes1 for their turnover. Our finding suggest that Fes1 is required for the degradation of misfolded proteins provides

an explanation for the accumulation of misfolded proteins in fes1Δ cells and the disturbed proteostasis that induces the heat-shock response. Fes1 releases misfolded proteins from Hsp70 After showing that misfolded DHFR proteins require Fes1 for their degrada-tion, we asked whether Fes1 promotes their release from Hsp70. We utilised

wild-type and fes1Δ cells, and monitored the constitutively expressed Ssa2 pro-tein level. Strikingly, upon immunoprecipitating DHFRmutC we found that Ssa2

was more associated in fes1Δ cells than in wild-type cells. Further, to determine whether the Fes1 associated with misfolded protein, we utilised DHRFmutC-FLAG in pull-down experiments. Surprisingly, Fes1 copurified with DHRFmutC-FLAG, but not with the natively folded protein DHFR-FLAG. This indicates that Fes1 interacts specifically with misfolded proteins, and that Fes1 promotes the release of misfolded proteins that are associated with Hsp70. We have summarized our findings regarding the functions of Fes1 and Hsp110 in controlling substrate release from Hsp70 in a graphical model (Figure 12). Briefly, the model distinguishes between the roles of NEFs played by Hsp110 and Fes1. Our results show that Fes1 is required for targeting Hsp70-associated

misfolded proteins for degradation. In comparison to fes1Δ cells, sse1Δ cells are slow-growing, unable to induce a strong heat shock response, and accumulate

misfolded proteins. These observations suggest that sse1Δ cells induce a general malfunction of Hsp70 system, and that these phenotypes are key in distinguish-ing Fes1 from Sse1. In conclusion, cells lacking Sse1 are more liable to undergo protein aggregation, and the aggregated proteins compete for the degradation pathway.

43

Figure 12: Model for the regulation of Hsp70 by NEF to determine sub-strate fate.

Hsp70

Hsp110

Fes1

Degradation

Folding

Substrate

ADP

Hsp11ATP

ADP

ADP

FoldF

P

ging

ATP

44

5.2 Study II 5.2.1 Aim Aim 2. Characterize Fes1 nucleo-cytoplasmic localisation, functional expression and regulation Study II describes how alternative splicing regulates the expression and func-tion of Fes1 in PQC by producing two splice isoforms. We have found that the FES1 locus extends beyond the originally proposed coding sequence to include a 3' intron, followed by a second exon. Transcriptional termination in the intron results in the expression of a short isoform known as “Fes1S”. In con-trast, excision of the intron extends the C-terminus to produce a long isoform known as “Fes1L”. While Fes1S is localised to the cytosol, Fes1L is actively targeted to the nucleus. The study provides information about the importance of the splice isoforms in PQC in the cytosol and nucleus. 5.2.2 Results and discussion FES1 transcripts undergo alternative splicing, resulting the expression of the Fes1S and Fes1L isoforms We examined the FES1 locus downstream of the previously identified ORF and found conserved sequence elements associated with an intron and a second exon. Sequencing of PCR products from reverse transcribed FES1 mRNA demonstrated that splicing indeed occurred, and that the transcripts extended beyond the previously identified ORF. FES1 splicing resulted in the expression of two isoforms with different C-terminal sequences. The spliced transcript encoded an isoform that was extended with twelve amino acids. The expressed isoforms were named “Fes1S” and “Fes1L”, with molecular weights predicted to be 32.6 kDa and 34.2 kDa, respectively. Fes1S is the predominant form, while Fes1L is expressed at lower levels. The genetic organisation of the FES1 locus and our results indicate that the alternative splicing is the consequence of competition between the activities of the splicing and polyadenylation machin-eries.

45

Fes1 isoform expression is modulated by heat stress Hsf1 controls the transcription of FES1 and thus may regulate the alternative splicing. Upon examination of FES1 mRNA levels during non-stressful and heat-shock conditions, we found that transcripts specific for Fes1S were prefer-entially induced during heat shock. Quantification of protein levels supported the suggestion of the preferential induction of Fes1S over Fes1L during heat shock. Our experiments also show that both isoforms are highly stable proteins and their turnover is undetectable. In summary, these observations suggest that the Fes1S isoform is the stress-inducible isoform. Fes1 isoforms have similar NEF activities We purified the Fes1 isoforms from bacteria and determined their NEF activi-ties using a stopped-flow instrument. Hsp70 (Ssa1) was incubated with MABA-ATP (a fluorescent ATP derivative) and was rapidly mixed with either Fes1S or Fes1L. The nucleotide release rates for the isoforms were 3.7 s-1 and 3.9 s-1, respectively. Our results indicate that the two isoforms have comparable NEF activities. Fes1S localises to the cytosol and Fes1L is actively targeted to the nucle-us Upon close examination of the extended C-terminal tail of Fes1L, we found a predicted nuclear localisation signal (NLS). The NLS motif was conserved between Fes1L homologues in other Saccharomycetaceae species. We examined the localisation of the Fes1 isoforms by fusing GFP to each protein. Fluores-cence microscopy revealed that Fes1S was localised mainly to the cytosol while Fes1L was targeted to the nucleus. Fes1L is the first Hsp70 NEF to be reported to actively localised to the nucleus. Fes1S is required for growth at elevated temperatures, and is required to maintain the repressed state of the heat-shock response We constructed genomically modified strains that expressed only Fes1S or Fes1L, in order to investigate which of the isoforms is crucial for Fes1 func-tion. The strains were subjected to phenotypic growth tests at elevated tem-

46

peratures, and also to toxic amino acid analogue canavanine. The Fes1S-expressing strain grew in a manner similar to that of the wild-type strain, whereas the Fes1L-expressing strain displayed clear growth defects. These re-sults support the suggestion that Fes1S is crucial for cell survival under proteo-toxic stress conditions. Study I had revealed that cells lacking Fes1 had an induced heat-shock response. We used transcriptional profiling to understand the differential heat shock response phenotypes associated with the Fes1 isoforms. We observed that wild-type and the constructed Fes1S-expressing strain matched closely, and

only four genes were differentially expressed. In contrast, the fes1Δ and the Fes1L-expressing strain displayed very distinct patterns, with a strong induction of the heat-shock response. Overall our results indicate that Fes1S maintains the heat-shock response repressed. Fes1S is an important factor required for the degradation of misfolded proteins We asked which of the isoforms is involved in the degradation of misfolded proteins in the cytosol and nucleus. We initially tested the misfolded protein Rpo41* and found that the protein was dependent on Fes1S, but not on Fes1L. We subsequently used misfolded protein Ste6*C, a misfolded protein that local-ises to the cytosol and requires mainly Ubr1 for its degradation. Ste6*C was

stabilised in fes1Δ cells and the degradation required Fes1S, but not Fes1L. Fi-

nally, we checked the degradation kinetics of ΔssPrA, vacuolar protease A that mislocalizes to the cytosol and misfolds after truncation of its signal sequence.

ΔssPrA is targeted to the nucleus for San1-dependent degradation. Again, the misfolded protein depended on Fes1S, but not Fes1L, for its degradation. Up-

on overexpression of Fes1L, degradation of Ste6*C and ΔssPrA was partially restored, probably as a consequence of an increased expression of Fes1L in the cytosol. We also analysed the nuclear misfolded model proteins Sed1*-GFP and Rad16*-GFP, which require San1 for their degradation. Neither of these sub-strates required Fes1 for their turnover. These results show that Fes1S is re-quired for the degradation of misfolded proteins in the cytosol. Strikingly, Fes1S plays a key role in regulating the proteotoxic effects of

misfolded proteins. Expression of Ste6*C and ΔssPrA in fes1Δ and in the Fes1L-expressing strain, but not in the wild-type and Fes1S strains, resulted in

47

strong growth inhibition. Thus, this observation supports the suggestion that Fes1 levels in the cytosol are critical to maintaining proteostasis during the production of proteotoxic misfolded proteins. We summarize our findings in a model that develops the concepts established in Study I (Figure 13). Briefly, when cytosolic Hsp70 encounters Fes1S, mis-folded protein bound by Hsp70 is released and is targeted for Ubr1-dependent and San1-dependent degradation. Fes1L, in contrast, resides in the nucleus and is not involved in the degradation of San1-dependent misfolded proteins. Cells lacking Fes1S are sensitive to the presence of misfolded proteins and induce the heat-shock response. The model also shows the crucial role of Fes1S in main-taining proteostasis, and the way in which it delivers the misfolded protein to Hsp70, leading to its degradation by the ubiquitin-proteasome system.

Figure 13: Model showing the localization of Fes1 isoforms and their regulation of Hsp70 function.

Hsp70Misfoldedprotein

ADP

Cytosol Nucleus

Fes1SATP

UPS degradation

ADP

Fes1LATP

UPS degradation

San1

Ubr1

egradationegradaegrada

NLS

HSPHSE

Hsf1

48

5.3 Study III 5.3.1 Aim Aim 3. Provide mechanistic insight into how Fes1 regulates Hsp70 This study elucidated the role of the flexible N-terminal domain (NTD) of Fes1, which plays a crucial role in the function of Fes1 and interacts with Hsp70. Studies I and II had shown that Fes1 plays a specialized role in mediat-ing the degradation of misfolded proteins in PQC. The activities beyond its function as an NEF required for this cellular function, however, needed to be defined. In this study, we unexpectedly found that the NTD is a domain that specifies the cellular function of Fes1. 5.3.2 Results and discussion Fes1 carries an extended and conserved N-terminal domain (NTD) that is required for in v ivo function Both splice isoforms of Fes1 carry conserved NTDs and armadillo domains. The Fes1 NTD has a length of 38 amino acids and, interestingly, carries two repeated motifs KLLQ at positions 1-9 and 25-38. The N-terminal domain ends with a glycine linker that connects to the armadillo domain. The conserva-tion of an NTD within the fungal Fes1 homologues suggests that Fes1 plays an important, conserved role.

We constructed a fes1ΔNT allele in which residues 2-34 of the NTD had been removed. Surprisingly, we found that even though the NTD is not part of the armadillo domain (which is the well-characterized Hsp70-interacting domain),

the NTD was required to complement fes1Δ growth phenotypes. Western anal-

ysis ruled out that the phenotypes of fes1ΔNT were the result of reduced ex-

pression levels of the truncated protein, since Fes1ΔNTD was expressed at slightly higher levels than wild-type protein. In the light of the very high stabil-ity of Fes1S, the slightly higher levels of expression are probably the result of an activated heat-shock response that induces the FES1 promoter in response to the non-complementation phenotype. We tested directly whether the heat-shock response was induced using a luciferase reporter based on NlucPEST

49

driven by a heat-shock responsive promoter. The fes1ΔNT was found to consti-tutively induce the heat-shock response. Next, we tested whether the NTD is required for the degradation of misfolded proteins. As in the case of the other phenotypes, the NTD was required for the degradation of misfolded protein

ΔssPrA. Results from all the above phenotypes indicate that the NTD is re-quired for the in vivo function of Fes1. The function of the NTD is transferable and the NTD is not required for the structural integrity of the armadillo domain The function of the NTD relates either to its physical interaction with the ar-madillo domain or to interactions with other factors. We fused the NTD to the BAG domain of Snl1 in order to test whether the NTD can function in the context of other NEFs that are not structurally related to Fes1. Surprisingly, the

chimeric protein suppressed fes1Δ growth phenotypes dependent on the NTD. This result indicates that the activities of the NTD are transferable and modu-lar, perhaps by interactions with other factors.

To analyze the biochemical functions, we purified Fes1 lacking its NTD (ΔNT) and Fes1S from Escherichia coli. First, we subjected the proteins to limited prote-olysis by trypsin to assess the compactness of the folded proteins. The NTD contains four potential cleavage sites for trypsin. SDS-PAGE analysis revealed

that, while ΔNT was a completely stable protein under the assay conditions, Fes1S rapidly lost its NTD by proteolysis. Thus, the NTD is a flexible domain attached to the compact armadillo domain. In parallel to these experiments, we tested the structural stability of Fes1S and

ΔNT by circular dichroism (CD) measurements. The measurements were per-formed with purified proteins incubated at temperatures from 20 oC to 80 oC.

The results show that both proteins have a high α-helical content and the

Fes1S and ΔNT have nearly the same melting temperatures: 55±4 oC and 53±3 oC, respectively. These results indicate that the NTD is an exposed flexible domain that does not affect the stability of the armadillo domain.

50

The NTD interacts with Hsp70 The observations described above led us to predict that the NTD is in direct contact with Hsp70, in parallel with the NEF interactions mediated by the ar-madillo domain. We used the LIMBO algorithm to find predicted Hsp70-binding sites in the Fes1 amino acid sequence. Notably, the NTD peptide 2-8 (EKLLQWS) was scored as a highly significant predicted peptide, which could be bound by Hsp70 as a substrate. This raises the possibility that the NTD functions as a substrate-like interaction domain with the SBD of Hsp70. We used an in vitro approach to test whether Fes1 NTD binds Hsp70. The iso-lated NTD was fused to GST and was incubated with purified Hsp70. Using glutathione chromatography, we observed that Hsp70 formed a complex with the NTD. Next, we asked whether the complex was sensitive to the presence of ATP; a hallmark substrate of Hsp70, as well as NEF-Hsp70 interactions. The interaction was indeed sensitive to ATP, as shown by the fact that at least a fraction of the bound protein was eluted by nucleotide addition. These results show that NTD-Hsp70 interactions are sensitive to the presence of nucleotides, presumably since the NTD binds the SBD of Hsp70. The observations described above lead us to propose the model shown in Fig-ure 14, in which the Fes1 isoform interacts with Hsp70, accompanied by the transition of ADP to ATP. When the armadillo domain interacts with the NBD of Hsp70, the NTD of Fes1 might extend its interaction to the SBD of Hsp70. ATP rebinding to Hsp70 leads to an open conformation, while the Fes1 iso-form might simultaneously leave the SBD. This indicates that NTD is a poten-tial substrate for Hsp70.

Figure 14: Model for Hsp70 and substrate interaction, indicating Fes1 interaction.

51

5.4 Study IV 5.4.1 Aim Aim 4. Streamlining the assembly of plasmids for recombinant protein expression in bacteria This study describes the development of a versatile vector (pSUMO-YHRC) that facilitates the single-step assembly of bacterial expression plasmids using yeast homologous recombination. The study develops a protocol for the rapid and highly efficient assembly of plasmids from single DNA fragments and up to seven DNA fragment. 5.4.2 Results and discussion The pSUMO-YHRC is an E. coli protein expression vector that carries an inducible T7 promoter. It stems from the standard pET24 vector, which has been modified to facilitate its propagation and selection in both bacterial and yeast. Briefly, we exploited our finding that the TEF promoter from Ashbya gossypii is bifunctional and enables the expression of the kanamycin-resistance gene in both yeast and bacteria. Introduced into pET24 together with a CEN/ARS fragment, this allows pSUMO-YHRC to be propagated and select-ed for in both species. The vector also carries the hexahistidine-tagged SUMO gene, which promotes the soluble expression of many proteins that are difficult to express. The SUMO moiety is easily removed by the SUMO protease Ulp1 (a protease that is highly active, specific and insensitive to buffer conditions) to yield the native protein. We have assembled a variety of expression plasmids using the pSUMO-YHRC vector backbone. Briefly, efficient cloning is achieved by cotransforming the crude PCR product with the restricted vector to yield G418-resistant yeast cells. After 2 days of growth, the yeast transformants are pooled, and the assembled plasmids are rescued by E. coli by selecting for kanamycin resistance. Verifica-tion of several clonings have shown that the overall efficiency for cloning single DNA fragments is 97%. We have used this approach for plasmid assembly to construct expression plasmids for many proteins, including plasmids that express a VHH single-chain antibody that binds GFP (NanobodyGFP).

52

The vector and cloning approach by homologous recombination in yeast is also highly efficient for the assembly of multiple DNA fragments in a single step. We tested multiple-fragment assembly for the 3.1 kb E. coli lacZ gene, which

encodes β-galactosidase. The lacZ gene was split into 2, 3 and 7 fragments. These were amplified by PCR and used for the methodology outlined above. Each fragment carried 50 bp of homology with its neighboring fragment. Upon plasmid isolation and verification, the 2, 3 and 7 fragments resulted in 90.7%,

88.3% and 85.7% clones, respectively, that expressed β-galactosidase. It is evi-dent that multiple-fragment assembly using pSUMO-YHRC is highly efficient, even with seven DNA fragments. In our approach, three sequential selections yield high efficiency. Firstly, selec-tion is that of G418-resistant yeast cells. Secondly, the DNA extraction from yeast specifically selects for plasmid DNA that can replicate in yeast. Finally, the transformation of E. coli selects only plasmids that can replicate and express kanamycin resistance in the presence of kanamycin. The approach requires minimal bench time, and it is a highly efficient and single-step protocol that can clone multiple fragments. Our approach has three advantages over the state-of-the art Gibson one-step isothermal in vitro recom-bination process. Firstly, it is highly efficient with even a large number of DNA fragments. Secondly, the protocol does not require any special proprietary reagents or kits, and finally, it works directly using crude PCR product.

53

6. Conclusions and outlook

I have studied in depth the conserved, abundant and stress-induced molecular chaperone Hsp70 and its regulation. Broadly, my thesis increases understanding of the molecular mechanisms that regulate the function of Hsp70 in the context of the larger proteostasis network, including both protein folding and degrada-tion machineries. Breakdown of the proteostasis network induces the accumu-lation of proteotoxic misfolded proteins, hampers cellular function, and is asso-ciated with disease. Hence, understanding the molecular mechanisms of the proteostasis network is essential. The functional diversification of multiple Hsp70 NEFs in the eukaryotic cyto-sol is poorly understood. In the yeast cytosol, three structurally distinct families of NEFs are represented by five proteins (Fes1L, Fes1S, Sse1, Sse2 and Snl1), and the work presented in this thesis adds to the small amount of knowledge previously available that gave insight into the activities that distinguish between their functions. For example, the Hsp110 family members Sse1 and Sse2 are also members of the Hsp70 superfamily, and have therefore generally been considered to function as chaperones. Experimental findings to support this suggestion are however, limited and controversial. Information about other NEFs is even more limited. Yet, the eukaryotic diversification and evolutionary conservation of multiple structurally distinct NEFs argue strongly for special-ized functions. In this respect, the findings of this thesis lend insight into the specific function of the Fes1 NEF, its subcellular localization in relation to its function, and a domain that may explain what distinguishes it from other NEFs. The basic concept of the model that was originally presented in Study I, ex-tended with spatial information in Study II, and with mechanistic details in Study III is that the NEFs interact with Hsp70 to affect the fate of associated protein substrates. The results presented here support the interpretation that the abundant NEFs of the Hsp110 class, Sse1 and Sse2, provide a bulk NEF function and support the protein-folding activities of Hsp70 (Raviol et al., 2006; Shorter, 2011; Xu et al., 2012). Indeed, the hypomorphic Hsp110 mutation

sse1Δ results in a general slow-growth phenotype and many defects associated with breakdown of the proteostasis network. Presumably, the removal of the abundant NEF Sse1 leads to impairment of general Hsp70 function. In con-trast, our results show that Fes1 has a much more specialized function, namely

54

to target Hsp70-associated misfolded proteins for degradation. Our main line of evidence to support this suggestion is the observation that Fes1 physically interacts with misfolded proteins that are associated with Hsp70. Fes1 is re-quired for misfolded protein degradation, cells lacking Fes1S accumulate mis-folded proteins and induce the proteostasis-regulated heat-shock response. Further support and a potential avenue for the mechanistic elucidation of the specialized function of Fes1 come from our on-going characterization of the Fes1 NTD presented in Study III. Thus, the NTD of Fes1 may partially explain the specialized function of Hsp70 NEFs. At present, our understanding of the role of Fes1 in PQC is based mainly on characterization of the phenotypes associated with fes1 mutations. A reconsti-tuted in vitro system would allow us to test the hypothesis that competition be-tween Fes1 and Sse1/Sse2 constitutes the main mechanism for substrate triage at Hsp70. If this competition is indeed the determinant of the fate of Hsp70-associated substrates, the question arises of how Sse1 promotes folding rather than degradation. Further characterization of Hsp110 function is clearly re-quired to understand the functional specialization of the cytosolic NEFs. The proposed role of Hsp110 in protein folding together with Hsp70 should be investigated by studying both de novo folding associated with translation and the reactivation of misfolded and damaged proteins, a process that is highly dependent on the powerful disaggregase Hsp104 in yeast. Indeed, Hsp70-Hsp40 together with Sse1, rather than Fes1, promotes the refolding of lucifer-ase aggregates (Shorter, 2011). In vitro evidence also suggests that Sse1 binds hydrophobic peptides (Shorter, 2011; Xu et al., 2012). Hence, the direct interac-tion of Sse1 with hydrophobic stretches of protein may maintain the non-natively folded proteins in a soluble state following NEF-induced release. Ac-cording to this scenario, Hsp110 coordinates nucleotide exchange with folding and guards proteins by shielding hydrophobic stretches that function as deg-rons and are recognized by ubiquitin ligases such as Ubr1 and San1. Our finding on alternative splicing of FES1 transcripts leads to the alternative expression of the nuclear-targeted Fes1L suggests that Fes1 participates in PQC in the nucleus, in addition to its activities in the cytosol. This hypothesis is supported by the similar NEF activities of both splice isoforms, and by the fact that both proteins carry the functionally critical NTD. The presence of the Hsp70 system and its function in PQC in the nucleus need to be better under-stood. Throughout the cell cycle, the proteasomes are known to localise to the

55

nucleus (Laporte et al., 2008), and in mammalian cells also Hsp70 targets the nucleus (Banski et al., 2010; Chu et al., 2001). These observations suggest that Hsp70 and the UPS are active in PQC in the nucleus. Indeed, misfolded cyto-plasmic proteins are targeted in yeast to the nucleus for proteolysis in an Hsp40-dependent manner (Park et al., 2013), and the presence of a dedicated nuclear San1 ubiquitin ligase lends further support to the importance of the nucleus in misfolded protein degradation (Gardner et al., 2005; Rosenbaum et al., 2011). These observations lead us to speculate that Fes1L executes its role to release misfolded proteins from Hsp70 in the nucleus in a manner analo-gous to the function of Fes1S in the cytosol. However, none of the limited set of nuclear misfolded model proteins that depend on San1 that we have tested so far depends on Fes1L for its turnover. Hence, there is a need to better un-derstand the spatial functional localisation of Fes1 activities. It has been reported by next generation mRNA sequencing that 4% of Saccha-romyces cerevisiae genes comprise introns, including FES1 as one of their hits (Nagalakshmi et al., 2008; Schreiber et al., 2015; Yassour et al., 2009). However, no studies have reported FES1 defined splice site and functional isoforms. Our findings show that Fes1 not only plays a role in protein quality control, but also in a rare splicing event. Fes1 undergoes alternative splicing through competition between a polyadenylation site that is proximal to the promoter and an intron. The Fes1 isoforms, Fes1S and Fes1L, have unique C-terminal, where Fes1S localises to the cytosol and Fes1L with NLS actively localised to the nucleus. Importantly, exon II of Fes1L is conserved within Saccharomycetaceae, and over-

expression of Fes1L can rescue the fes1Δ phenotype. These results suggest that Fes1L plays an important role. Hence, it is important to study the role of Fes1 splicing during stress, and to study the components involved in regulating Fes1 splicing. Our studies in this thesis define functions of the NTD, and the work reported in this thesis has revealed continue. We have yet to show that the ATP-sensitive interactions of the NTD actually play a role in its in vivo function. Future work may involve more detailed mutational analysis of the NTD and more extensive in vitro characterization of the domain. Direct competition experiments be-tween the NTD and known Hsp70 substrate peptides would clearly be im-portant to test the hypothesis of a substrate-like binding of the NTD. Curious-ly, the NTD is also the domain that is post-translationally modified in Fes1 (Albuquerque et al., 2008). The only phosphorylation that has been identified is

56

on serine twelve amino acid. Thus, more work is required to elucidate the func-tion and regulation of the NTD, and how the molecular mechanism of Fes1 differs from those of other NEFs. Hence, these studies on NEFs provide in-sight on their function and how they regulate Hsp70 function.

57

7. Sammanfattning på svenska Proteinveckning är den process där polypeptider veckas till sina nativa struk-turer. Ogynnsamma miljöförhållanden och vissa genetiska anlag inverkar nega-tivt på veckningsprocessen och ökar därför produktionen av giftiga felveckade proteiner. Felveckade proteiner identifieras och avlägsnas från cellen genom en kvalitetskontrollprocess. I denna process spelar molekylära chaperoner ur Hsp70-familjen en nyckelroll genom att direkt identifiera felveckade proteiner och medverka till deras avlägsnande. Hsp70:s funktion bygger på samspel med en mängd kofaktorer som reglerar dess katalytiska ATPasaktivitet. I denna avhandling har jag använt mig av en kombination av metoder från jästgenetik, cellbiologi och biokemi för att experimentellt få en inblick i hur Hsp70:s funkt-ion regleras av kofaktorer som accelererar nukleotidutbyte, s. k. nukleotidutby-tesfaktorer. Studie I visar att nukleotidutbytesfaktorn Fes1 är nödvändig för kvalitetskontroll av proteiner i cytosolen. Våra genetiska studier visar att Fes1:s aktivitet krävs för att felveckade proteiner i komplex med Hsp70 ska spjälkas av ubiquitin-proteasomsystemet. Mer specifikt så främjar Fes1 en fysiskt förbin-delse mellan dessa komplex och ubiquitinyleringsenzymet Ubr1 som i sin tur sänder det felveckade proteinet för spjälkning i proteasomen. De observerade konsekvenserna av att genetisk inaktivera Fes1 är att felveckade proteiner inte spjälkas utan istället upplagras som proteinaggregat samt aktiverar ett uråldrigt genetiskt stressprogram. Sammantaget visar de experimentalla resultaten att Fes1 sänder felveckade proteiner till spjälkning genom att släppa loss dem från Hsp70. Studie II visar ett ovanligt fall av alternativ splitsning av FES1-transkripten som leder till att de två isoformerna Fes1S och Fes1L uttrycks. De båda isoformerna är fullt aktiva nucleotidsutbytesfaktorer men har olika lokali-sering i cellen. Fes1S lokaliserar till cytosolen och krävs för spjälkning av fel-veckade proteiner. Fes1L däremot lokaliserar till cellkärnan och utgör det första exemplet i jästceller på en nuklotidutbytesfaktor i cellkärnan. Upptäckten av isofomer av en nukleotidutbytesfaktor har betydelse för hur vi ser på mekan-ismen bakom kvalitetkontroll av proteiner. Studie III beskriver mekanismen som Fes1 använder för att kontrollera Hsp70:s funktion. Fes1 bär på en evolut-ionärt konserverad N-terminal domän som saknar fast struktur, är modulär och krävs för att Fes1 ska fungera i cellen. Domänen bildar ATP-känsliga komplex med Hsp70, vilket tyder på att den binder i samma säte som felveckade protei-ner och därmed påverkar deras interaktion med Hsp70. Studie IV beskriver en ny metod baserad på homolog rekombination i jäst som underlättar byggandet av plasmider för genuttryck i bakterier. Upptäckerna i avhandlingen ger vid handen att Fes1 spelar en nyckelroll i de mekanismer i cellen som utför kvali-tetskontroll av proteiner och sänder felveckade proteiner för spjälkning i prote-asomen. I ett bredare perspektiv ger dessa fynd viktig information som krävs för att utveckla modeller som beskriver hur Hsp70:s funktion regleras av olika kofaktorer för att delta i veckning och spjälkning av proteiner.

58

8. Acknowledgements I am grateful to Dr. Claes Andréasson my guru (supervisor), glad to work as your first PhD student. Thankful for giving me a wonderful opportunity to work on this project. Your patience, guidance and freedom to work in this project helped me a lot to achieve many experimental and scientific writing skills. I would like to thank my co-supervisor Prof. Per Ljungdahl, for discussions and guiding throughout my studies for the improvement. I will take this opportunity to thank researcher Prof. Roger Karlsson, Prof. Eva Severinson and Prof. Ann-Kristin Östlund Farrants for creating good research environment around. This always helped me to keep up the good work. I also thank IFSU facility and thank Stina H for helping me to acquire knowledge on microscopy. Also would like to thank Prof. Martin Ott, and his group members DBB, Stockholm University for your great inputs on my project during the discus-sions in yeast lab meetings. I would like to thank my collaborators Prof. Jürgen Dohmen, University of Cologne, Cologne, Germany and Asst. Prof. Marc Friedländer, Stockholm University, Stockholm for the collaboration to execute my projects and your contributions are very much valuable. I would like to thank Prof. Lena Mä-ler and Prof. Peter Brzezinski, DBB, Stockholm University, for providing opportunity to carry out experiments in your laboratory. Jobst L for you kindness and helping me with the CD spectra very much in time. I would like to thank administrative staff for helping me during my entire studies at MBW-WGI, Stockholm University. Special thanks to my lab mates Jayasankar, Anna and Ganapathi for all your support, we were not just colleagues also good friends. Jayasankar for the contribution to my project and you are a great teammate. Thanks Ga-napathi - ubiquitin expert in our lab, you have always added good inputs on my project. Thank Anna my Swedish tutor and being contributing to my work and for baking cakes for us. Glad to be with you all, it was really great fun working and the moments are cherishable. Thanks for your patience and taking your time on reading my thesis. Thanks to Mats for your scientific inputs on my project. Finally thank Fabian and João for all the conversa-tions together.

59

Thanks to Kicki Ryman for being so kind to me and helping whenever re-quired. Thanks to past and present members of Per Ljungdahl group for all the fun together and not making me feel lonely during the beginning of my studies. Thanks to Antonio, Andreas, Fitz and Marina for all crazy talks and joy together. Special thanks to Antonio for you kindness and your scientific thoughts and the experience in teaching together. It was pleasure to work with my present colleagues Misa, Yuan, Anna R, Jaclyn and past colleagues Ming, Natalija, Anna V, Steffi, Fathemehm, Sara, Matthias, Deike for all the fun and discussions during corridor meetings. I always enjoyed playing badminton with Jayashankar, Toni, Matthias and Ming. Within a short span of time, it was nice to interact with Sabarina Büttner and group mem-bers and thank you for the help and discussions. Thanks to my F4 north cor-ridor friends for making it more productive environment. To my friends and family, you should know that your support and encour-agement was worth more than I can express on paper. I am really indebted for all those who have given me their friendship, stood by me in my odd times and provided me lot of support and practical help. Personally, I would like to thank my family: my uncle Shivalingappa, Aunt Ratnamma, father Chandappa Gowda, mother Yashodamma for supporting me throughout the years, financially, practically and with moral support. I would like to dedicate my thesis to my uncle Mr. Rudregowda S and his wife, my aunt Shakunthala Y P who actually supported me to reach this goal and would not had happened without their support and encouragement. I am always grateful to Harsha anna - Geetha akka ; Chaitra akka - Arun anna for your support and encouragement. You and your family have always stood along with me in all my accomplishments and have encouraged me. Special thanks to my sisters family Sunitha – Mallikarjun, Sudha – Somanath for your immense support all the time and glad to get great sisters like you. You both are my real support and have given me unconditional love throughout my life and studies. Special thanks to my wife Divya for love, affection and huge support have helped me to accomplish this goal. And thanks to Bhavya, my uncle Shival-ingappa and my aunt Ambika for your affection and support. My extended thanks to Eshwarappa and family; Late Nagendrappa and his family; C Rudrappa and his family and LG family at Basavapatna for your love and affection and each one of your family members have directly or indirectly contributed to achieve this.

60

My thanks to Harisha and Shruthi for all the fun together and helping me in all kinds. Harisha you were with me in all my ups and downs. Your thoughts and positive energy are exceptional. I am glad to get a great friend like you and you are very much supportive in professional and personal life and I can never forget that. Thanks to Sagar Manoli for introducing me to the world of Stockholm and all the help and support together with Nirmala. Thanks Samarth for all the fun and enjoyment together and so many years of friendship being together and Sonam is the best match for you in all aspect. Thanks to our Stockholm friends Harsha-Shreya; Jayanth-Vinutha; Swaroop-Praghna for all the fun at get togethers and are never forgettable. My friends Vinay, Srikanth, Vikaram, Sundar, Vineeth, Ganesh and Ma-hesh whom I met in Stockholm had lot of fun. Your warm friendship and was great being with you guys and we have many unforgettable moments. My short time at Uppsala made good Uppsalites. Chethan my best friend and for being so helpful at difficult times and all the good memories staying together are always exceptional, Shwetha is your perfect life partner. Nikhil and Lakshmi - thank you so much for the fun together and I am really grate-ful for the hospitality. My buddy Raghuveer and Madhu being a kind friends and it was nice being with you both. Not but not least, I would like to thank my all relatives and friends who di-rectly or indirectly supported me throughout my studies.

61

9. References Abrams, J.L., J. Verghese, P.A. Gibney, and K.A. Morano. 2014.

Hierarchical functional specificity of cytosolic heat shock protein 70 (Hsp70) nucleotide exchange factors in yeast. The Journal of biological chemistry. 289:13155-13167.

Agashe, V.R., M.C. Shastry, and J.B. Udgaonkar. 1995. Initial hydrophobic collapse in the folding of barstar. Nature. 377:754-757.

Alberti, S., K. Bohse, V. Arndt, A. Schmitz, and J. Hohfeld. 2004. The cochaperone HspBP1 inhibits the CHIP ubiquitin ligase and stimulates the maturation of the cystic fibrosis transmembrane conductance regulator. Mol Biol Cell. 15:4003-4010.

Alberti, S., C. Esser, and J. Hohfeld. 2003. BAG-1--a nucleotide exchange factor of Hsc70 with multiple cellular functions. Cell Stress Chaperones. 8:225-231.

Albuquerque, C.P., M.B. Smolka, S.H. Payne, V. Bafna, J. Eng, and H. Zhou. 2008. A multidimensional chromatography technology for in-depth phosphoproteome analysis. Molecular & cellular proteomics : MCP. 7:1389-1396.

Anckar, J., and L. Sistonen. 2011. Regulation of HSF1 Function in the Heat Stress Response: Implications in Aging and Disease. Annual Review of Biochemistry. 80:1089-1115.

Andréasson, C., J. Fiaux, H. Rampelt, S. Druffel-Augustin, and B. Bukau. 2008. Insights into the structural dynamics of the Hsp110–Hsp70 interaction reveal the mechanism for nucleotide exchange activity. Proceedings of the National Academy of Sciences. 105:16519-16524.

Anfinsen, C.B. 1973. Principles that govern the folding of protein chains. Science. 181:223-230.

Ares, M., L. Grate, and M.H. Pauling. 1999. A handful of intron-containing genes produces the lion's share of yeast mRNA. Rna. 5:1138-1139.

Ast, G. 2004. How did alternative splicing evolve? Nature reviews. Genetics. 5:773-782.

Balch, W.E., R.I. Morimoto, A. Dillin, and J.W. Kelly. 2008. Adapting Proteostasis for Disease Intervention. Science. 319:916-919.

Baldwin, R.L., and G.D. Rose. 1999. Is protein folding hierarchic? II. Folding intermediates and transition states. Trends Biochem Sci. 24:77-83.

Banski, P., H. Mahboubi, M. Kodiha, S. Shrivastava, C. Kanagaratham, and U. Stochaj. 2010. Nucleolar targeting of the chaperone hsc70 is regulated by stress, cell signaling, and a composite targeting signal which is controlled by autoinhibition. The Journal of biological chemistry. 285:21858-21867.

Bartel, B., I. Wunning, and A. Varshavsky. 1990. The recognition component of the N-end rule pathway. The EMBO journal. 9:3179-3189.

62

Baxter, B.K., and E.A. Craig. 1998. Isolation of UBP3, encoding a de-ubiquitinating enzyme, as a multicopy suppressor of a heat-shock mutant strain of S. cerevisiae. Current genetics. 33:412-419.

Becker, J., and E.A. Craig. 1994. Heat-shock proteins as molecular chaperones. European Journal of Biochemistry. 219:11-23.

Bengtson, M.H., and C.A. Joazeiro. 2010. Role of a ribosome-associated E3 ubiquitin ligase in protein quality control. Nature. 467:470-473.

Boorstein, W., T. Ziegelhoffer, and E. Craig. 1994. Molecular evolution of the HSP70 multigene family. J Mol Evol. 38:1-17.

Bracher, A., and J. Verghese. 2015. The nucleotide exchange factors of Hsp70 molecular chaperones. Frontiers in molecular biosciences. 2:10.

Brauer, M.J., C. Huttenhower, E.M. Airoldi, R. Rosenstein, J.C. Matese, D. Gresham, V.M. Boer, O.G. Troyanskaya, and D. Botstein. 2008. Coordination of growth rate, cell cycle, stress response, and metabolic activity in yeast. Mol Biol Cell. 19:352-367.

Buchberger, A., B. Bukau, and T. Sommer. 2010. Protein quality control in the cytosol and the endoplasmic reticulum: brothers in arms. Molecular cell. 40:238-252.

Cattaneo, M., R. Dominici, M. Cardano, G. Diaferia, E. Rovida, and I. Biunno. 2012. Molecular chaperones as therapeutic targets to counteract proteostasis defects. Journal of cellular physiology. 227:1226-1234.

Cheetham, M.E., and A.J. Caplan. 1998. Structure, function and evolution of DnaJ: conservation and adaptation of chaperone function. Cell Stress Chaperones. 3:28-36.

Chu, A., N. Matusiewicz, and U. Stochaj. 2001. Heat-induced nuclear accumulation of hsc70s is regulated by phosphorylation and inhibited in confluent cells. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 15:1478-1480.

Cohen, F.E., and J.W. Kelly. 2003. Therapeutic approaches to protein-misfolding diseases. Nature. 426:905-909.

Craig, E.A., and K. Jacobsen. 1984. Mutations of the heat inducible 70 kilodalton genes of yeast confer temperature sensitive growth. Cell. 38:841-849.

Craig, E.A., and K. Jacobsen. 1985. Mutations in cognate genes of Saccharomyces cerevisiae hsp70 result in reduced growth rates at low temperatures. Molecular and cellular biology. 5:3517-3524.

Cuenca-Bono, B., V. García-Molinero, P. Pascual-García, H. Dopazo, A. Llopis, J. Vilardell, and S. Rodríguez-Navarro. 2011. SUS1 introns are required for efficient mRNA nuclear export in yeast. Nucleic Acids Research. 39:8599-8611.

Cuervo, A. 2004. Autophagy: Many paths to the same end. Mol Cell Biochem. 263:55-72.

63

Cuervo, A.M., L. Stefanis, R. Fredenburg, P.T. Lansbury, and D. Sulzer. 2004. Impaired Degradation of Mutant α-Synuclein by Chaperone-Mediated Autophagy. Science. 305:1292-1295.

Cyr, D.M., T. Langer, and M.G. Douglas. 1994. DnaJ-like proteins: molecular chaperones and specific regulators of Hsp70. Trends Biochem Sci. 19:176-181.

Demartino, G.N., and T.G. Gillette. 2007. Proteasomes: machines for all reasons. Cell. 129:659-662.

Deshaies, R.J., and C.A. Joazeiro. 2009. RING domain E3 ubiquitin ligases. Annu Rev Biochem. 78:399-434.

Dobson, C.M. 2004. Principles of protein folding, misfolding and aggregation. In Seminars in cell & developmental biology. Vol. 15. Elsevier. 3-16.

Dobson, C.M., and M. Karplus. 1999. The fundamentals of protein folding: bringing together theory and experiment. Current opinion in structural biology. 9:92-101.

Doyle, S.M., O. Genest, and S. Wickner. 2013. Protein rescue from aggregates by powerful molecular chaperone machines. Nature reviews. Molecular cell biology. 14:617-629.

Dragovic, Z., S.A. Broadley, Y. Shomura, A. Bracher, and F.U. Hartl. 2006a. Molecular chaperones of the Hsp110 family act as nucleotide exchange factors of Hsp70s. The EMBO journal. 25:2519-2528.

Dragovic, Z., Y. Shomura, N. Tzvetkov, F.U. Hartl, and A. Bracher. 2006b. Fes1p acts as a nucleotide exchange factor for the ribosome-associated molecular chaperone Ssb1p. Biological chemistry. 387:1593-1600.

Easton, D.P., Y. Kaneko, and J.R. Subjeck. 2000. The hsp110 and Grp170 stress proteins: newly recognized relatives of the Hsp70s. Cell stress & chaperones. 5:276.

Eisele, F., and D.H. Wolf. 2008. Degradation of misfolded protein in the cytoplasm is mediated by the ubiquitin ligase Ubr1. FEBS letters. 582:4143-4146.

Engebrecht, J., K. Voelkel-Meiman, and G.S. Roeder. 1991. Meiosis-specific RNA splicing in yeast. Cell. 66:1257-1268.

Fan, C.-Y., S. Lee, and D.M. Cyr. 2003. Mechanisms for regulation of Hsp70 function by Hsp40. Cell stress & chaperones. 8:309.

Fang, N.N., A.H. Ng, V. Measday, and T. Mayor. 2011. Hul5 HECT ubiquitin ligase plays a major role in the ubiquitylation and turnover of cytosolic misfolded proteins. Nat Cell Biol. 13:1344-1352.

Fersht, A.R., and V. Daggett. 2002. Protein folding and unfolding at atomic resolution. Cell. 108:573-582.

Finley, D., H.D. Ulrich, T. Sommer, and P. Kaiser. 2012. The Ubiquitin–Proteasome System of Saccharomyces cerevisiae. Genetics. 192:319-360.

Gallagher, P.S., S.V. Clowes Candadai, and R.G. Gardner. 2014. The requirement for Cdc48/p97 in nuclear protein quality control

64

degradation depends on the substrate and correlates with substrate insolubility. Journal of cell science. 127:1980-1991.

Gardner, R.G., Z.W. Nelson, and D.E. Gottschling. 2005. Degradation-mediated protein quality control in the nucleus. Cell. 120:803-815.

Gautschi, M., H. Lilie, U. Fünfschilling, A. Mun, S. Ross, T. Lithgow, P. Rücknagel, and S. Rospert. 2001. RAC, a stable ribosome-associated complex in yeast formed by the DnaK-DnaJ homologs Ssz1p and zuotin. Proceedings of the National Academy of Sciences. 98:3762-3767.

Gautschi, M., A. Mun, S. Ross, and S. Rospert. 2002. A functional chaperone triad on the yeast ribosome. Proceedings of the National Academy of Sciences of the United States of America. 99:4209-4214.

Geiler-Samerotte, K.A., M.F. Dion, B.A. Budnik, S.M. Wang, D.L. Hartl, and D.A. Drummond. 2011. Misfolded proteins impose a dosage-dependent fitness cost and trigger a cytosolic unfolded protein response in yeast. Proceedings of the National Academy of Sciences of the United States of America. 108:680-685.

Ghaemmaghami, S., W.K. Huh, K. Bower, R.W. Howson, A. Belle, N. Dephoure, E.K. O'Shea, and J.S. Weissman. 2003. Global analysis of protein expression in yeast. Nature. 425:737-741.

Gietz, R.D., and R.A. Woods. 2002. Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods in enzymology. 350:87-96.

Goeckeler, J.L., A. Stephens, P. Lee, A.J. Caplan, and J.L. Brodsky. 2002. Overexpression of Yeast Hsp110 Homolog Sse1p Suppressesydj1-151 Thermosensitivity and Restores Hsp90-dependent Activity. Molecular Biology of the Cell. 13:2760-2770.

Goffeau, A., B.G. Barrell, H. Bussey, R.W. Davis, B. Dujon, H. Feldmann, F. Galibert, J.D. Hoheisel, C. Jacq, M. Johnston, E.J. Louis, H.W. Mewes, Y. Murakami, P. Philippsen, H. Tettelin, and S.G. Oliver. 1996. Life with 6000 genes. Science. 274:546, 563-547.

Gottesman, S., S. Wickner, and M.R. Maurizi. 1997. Protein quality control: triage by chaperones and proteases. Genes & Development. 11:815-823.

Groll, M., M. Bochtler, H. Brandstetter, T. Clausen, and R. Huber. 2005. Molecular machines for protein degradation. Chembiochem : a European journal of chemical biology. 6:222-256.

Grund, S.E., T. Fischer, G.G. Cabal, O. Antúnez, J.E. Pérez-Ortín, and E. Hurt. 2008. The inner nuclear membrane protein Src1 associates with subtelomeric genes and alters their regulated gene expression. The Journal of Cell Biology. 182:897-910.

Hahn, J.S., Z. Hu, D.J. Thiele, and V.R. Iyer. 2004. Genome-wide analysis of the biology of stress responses through heat shock transcription factor. Molecular and Cellular Biology. 24:5249-5256.

Hanna, J., N.A. Hathaway, Y. Tone, B. Crosas, S. Elsasser, D.S. Kirkpatrick, D.S. Leggett, S.P. Gygi, R.W. King, and D. Finley. 2006.

65

Deubiquitinating enzyme Ubp6 functions noncatalytically to delay proteasomal degradation. Cell. 127:99-111.

Hartl, F.U., A. Bracher, and M. Hayer-Hartl. 2011. Molecular chaperones in protein folding and proteostasis. Nature. 475:324-332.

Hartl, F.U., and M. Hayer-Hartl. 2002. Molecular chaperones in the cytosol: from nascent chain to folded protein. Science. 295:1852-1858.

Heck, J.W., S.K. Cheung, and R.Y. Hampton. 2010. Cytoplasmic protein quality control degradation mediated by parallel actions of the E3 ubiquitin ligases Ubr1 and San1. Proceedings of the National Academy of Sciences of the United States of America. 107:1106-1111.

Hershko, A. 1996. Lessons from the discovery ofthe ubiquitin system. Trends in Biochemical Sciences. 21:445-449.

Hershko, A., E. Eytan, A. Ciechanover, and A.L. Haas. 1982. Immunochemical analysis of the turnover of ubiquitin-protein conjugates in intact cells. Relationship to the breakdown of abnormal proteins. The Journal of biological chemistry. 257:13964-13970.

Hipp, M.S., S.H. Park, and F.U. Hartl. 2014. Proteostasis impairment in protein-misfolding and -aggregation diseases. Trends in cell biology. 24:506-514.

Ho, A.K., G.A. Raczniak, E.B. Ives, and S.R. Wente. 1998. The integral membrane protein Snl1p is genetically linked to yeast nuclear pore complex function. Molecular biology of the cell. 9:355-373.

Hochstrasser, M. 1995. Ubiquitin, proteasomes, and the regulation of intracellular protein degradation. Current Opinion in Cell Biology. 7:215-223.

Hossain, M.A., C.M. Rodriguez, and T.L. Johnson. 2011. Key features of the two-intron Saccharomyces cerevisiae gene SUS1 contribute to its alternative splicing. Nucleic Acids Research. 39:8612-8627.

Huh, W.K., J.V. Falvo, L.C. Gerke, A.S. Carroll, R.W. Howson, J.S. Weissman, and E.K. O'Shea. 2003. Global analysis of protein localization in budding yeast. Nature. 425:686-691.

Jones, G., Y. Song, S. Chung, and D.C. Masison. 2004. Propagation of Saccharomyces cerevisiae [PSI+] prion is impaired by factors that regulate Hsp70 substrate binding. Molecular and Cellular Biology. 24:3928-3937.

Juneau, K., M. Miranda, M.E. Hillenmeyer, C. Nislow, and R.W. Davis. 2006. Introns regulate RNA and protein abundance in yeast. Genetics. 174:511-518.

Juneau, K., C. Nislow, and R.W. Davis. 2009. Alternative splicing of PTC7 in Saccharomyces cerevisiae determines protein localization. Genetics. 183:185-194.

Kabani, M., J.M. Beckerich, and J.L. Brodsky. 2002a. Nucleotide exchange factor for the yeast Hsp70 molecular chaperone Ssa1p. Molecular and Cellular Biology. 22:4677-4689.

66

Kabani, M., and C.N. Martineau. 2008. Multiple hsp70 isoforms in the eukaryotic cytosol: mere redundancy or functional specificity? Current genomics. 9:338.

Kabani, M., C. McLellan, D.A. Raynes, V. Guerriero, and J.L. Brodsky. 2002b. HspBP1, a homologue of the yeast Fes1 and Sls1 proteins, is an Hsc70 nucleotide exchange factor. FEBS letters. 531:339-342.

Kabbage, M., and M.B. Dickman. 2008. The BAG proteins: a ubiquitous family of chaperone regulators. Cellular and molecular life sciences : CMLS. 65:1390-1402.

Kaganovich, D., R. Kopito, and J. Frydman. 2008. Misfolded proteins partition between two distinct quality control compartments. Nature. 454:1088-1095.

Kampinga, H.H., and E.A. Craig. 2010. The HSP70 chaperone machinery: J proteins as drivers of functional specificity. Nature reviews. Molecular cell biology. 11:579-592.

Kettern, N., M. Dreiseidler, R. Tawo, and J. Hohfeld. 2010. Chaperone-assisted degradation: multiple paths to destruction. Biological chemistry. 391:481-489.

Kim, H.-K., R.-R. Kim, J.-H. Oh, H. Cho, A. Varshavsky, and C.-S. Hwang. 2014. The N-Terminal Methionine of Cellular Proteins as a Degradation Signal. Cell. 156:158-169.

Kim, Y.E., M.S. Hipp, A. Bracher, M. Hayer-Hartl, and F.U. Hartl. 2013. Molecular chaperone functions in protein folding and proteostasis. Annu Rev Biochem. 82:323-355.

Koplin, A., S. Preissler, Y. Ilina, M. Koch, A. Scior, M. Erhardt, and E. Deuerling. 2010. A dual function for chaperones SSB–RAC and the NAC nascent polypeptide–associated complex on ribosomes. The Journal of Cell Biology. 189:57-68.

Kraft, C., F. Reggiori, and M. Peter. 2009. Selective types of autophagy in yeast. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 1793:1404-1412.

Kryndushkin, D., and R.B. Wickner. 2007. Nucleotide exchange factors for Hsp70s are required for [URE3] prion propagation in Saccharomyces cerevisiae. Mol Biol Cell. 18:2149-2154.

Kumar, N., D. Gaur, D.C. Masison, and D. Sharma. 2014. The BAG homology domain of Snl1 cures yeast prion [URE3] through regulation of Hsp70 chaperones. G3. 4:461-470.

Labbadia, J., and R.I. Morimoto. 2015. The biology of proteostasis in aging and disease. Annu Rev Biochem. 84:435-464.

Laporte, D., B. Salin, B. Daignan-Fornier, and I. Sagot. 2008. Reversible cytoplasmic localization of the proteasome in quiescent yeast cells. J Cell Biol. 181:737-745.

Laskey, R.A., B.M. Honda, A.D. Mills, and J.T. Finch. 1978. Nucleosomes are assembled by an acidic protein which binds histones and transfers them to DNA. Nature. 275:416-420.

67

Li, J., X. Qian, and B. Sha. 2009. Heat shock protein 40: structural studies and their functional implications. Protein and peptide letters. 16:606.

Liu, Q., and W.A. Hendrickson. 2007. Insights into Hsp70 Chaperone Activity from a Crystal Structure of the Yeast Hsp110 Sse1. Cell. 131:106-120.

Mager, W.H., and A. De Kruijff. 1995. Stress-induced transcriptional activation. Microbiological reviews. 59:506-531.

Malinovska, L., S. Kroschwald, M.C. Munder, D. Richter, and S. Alberti. 2012. Molecular chaperones and stress-inducible protein-sorting factors coordinate the spatiotemporal distribution of protein aggregates. Mol Biol Cell. 23:3041-3056.

Martinez-Pastor, M.T., G. Marchler, C. Schuller, A. Marchler-Bauer, H. Ruis, and F. Estruch. 1996. The Saccharomyces cerevisiae zinc finger proteins Msn2p and Msn4p are required for transcriptional induction through the stress response element (STRE). The EMBO journal. 15:2227-2235.

Mayer, M., and B. Bukau. 2005. Hsp70 chaperones: cellular functions and molecular mechanism. Cellular and Molecular Life Sciences. 62:670-684.

McClellan, A.J., M.D. Scott, and J. Frydman. 2005a. Folding and quality control of the VHL tumor suppressor proceed through distinct chaperone pathways. Cell. 121:739-748.

McClellan, A.J., S. Tam, D. Kaganovich, and J. Frydman. 2005b. Protein quality control: chaperones culling corrupt conformations. Nature cell biology. 7:736-741.

Miller, G., and R. Mittler. 2006. Could heat shock transcription factors function as hydrogen peroxide sensors in plants? Annals of botany. 98:279-288.

Miller, S.B., C.T. Ho, J. Winkler, M. Khokhrina, A. Neuner, M.Y. Mohamed, D.L. Guilbride, K. Richter, M. Lisby, E. Schiebel, A. Mogk, and B. Bukau. 2015. Compartment-specific aggregases direct distinct nuclear and cytoplasmic aggregate deposition. The EMBO journal. 34:778-797.

Mizushima, N., and D.J. Klionsky. 2007. Protein Turnover Via Autophagy: Implications for Metabolism*. Annu. Rev. Nutr. 27:19-40.

Morimoto, R.I. 1993. Cells in stress: transcriptional activation of heat shock genes. SCIENCE-NEW YORK THEN WASHINGTON-. 259:1409-1409.

Mortimer, R.K., and J.R. Johnston. 1986. Genealogy of principal strains of the yeast genetic stock center. Genetics. 113:35-43.

Mukai, H., T. Kuno, H. Tanaka, D. Hirata, T. Miyakawa, and C. Tanaka. 1993a. Isolation and characterization of SSE1 and SSE2, new members of the yeast HSP70 multigene family. Gene. 132:57-66.

Mukai, H., T. Kuno, H. Tanaka, D. Hirata, T. Miyakawa, and C. Tanaka. 1993b. Isolation and characterization of SSE1 and SSE2, new members of the yeast HSP70 multigene family. Gene. 132:57.

68

Musgrove, J.E., and R.J. Ellis. 1986. The Rubisco Large Subunit Binding Protein. Philosophical Transactions of the Royal Society of London. B, Biological Sciences. 313:419-428.

Nagalakshmi, U., Z. Wang, K. Waern, C. Shou, D. Raha, M. Gerstein, and M. Snyder. 2008. The transcriptional landscape of the yeast genome defined by RNA sequencing. Science. 320:1344-1349.

Nillegoda, N.B., J. Kirstein, A. Szlachcic, M. Berynskyy, A. Stank, F. Stengel, K. Arnsburg, X. Gao, A. Scior, R. Aebersold, D.L. Guilbride, R.C. Wade, R.I. Morimoto, M.P. Mayer, and B. Bukau. 2015. Crucial HSP70 co-chaperone complex unlocks metazoan protein disaggregation. Nature. 524:247-251.

Nillegoda, N.B., M.A. Theodoraki, A.K. Mandal, K.J. Mayo, H.Y. Ren, R. Sultana, K. Wu, J. Johnson, D.M. Cyr, and A.J. Caplan. 2010. Ubr1 and Ubr2 function in a quality control pathway for degradation of unfolded cytosolic proteins. Mol Biol Cell. 21:2102-2116.

Normington, K., K. Kohno, Y. Kozutsumi, M.J. Gething, and J. Sambrook. 1989. S. cerevisiae encodes an essential protein homologous in sequence and function to mammalian BiP. Cell. 57:1223-1236.

Oling, D., F. Eisele, K. Kvint, and T. Nystrom. 2014. Opposing roles of Ubp3-dependent deubiquitination regulate replicative life span and heat resistance. The EMBO journal. 33:747-761.

Parenteau, J., M. Durand, S. Veronneau, A.A. Lacombe, G. Morin, V. Guerin, B. Cecez, J. Gervais-Bird, C.S. Koh, D. Brunelle, R.J. Wellinger, B. Chabot, and S. Abou Elela. 2008. Deletion of many yeast introns reveals a minority of genes that require splicing for function. Mol Biol Cell. 19:1932-1941.

Park, S.-H., Y. Kukushkin, R. Gupta, T. Chen, A. Konagai, Mark S. Hipp, M. Hayer-Hartl, and F.U. Hartl. 2013. PolyQ Proteins Interfere with Nuclear Degradation of Cytosolic Proteins by Sequestering the Sis1p Chaperone. Cell. 154:134-145.

Park, S.H., N. Bolender, F. Eisele, Z. Kostova, J. Takeuchi, P. Coffino, and D.H. Wolf. 2007. The cytoplasmic Hsp70 chaperone machinery subjects misfolded and endoplasmic reticulum import-incompetent proteins to degradation via the ubiquitin-proteasome system. Mol Biol Cell. 18:153-165.

Parsell, D.A., and R.T. Sauer. 1989. Induction of a heat shock-like response by unfolded protein in Escherichia coli: dependence on protein level not protein degradation. Genes Dev. 3:1226-1232.

Peng, J., D. Schwartz, J.E. Elias, C.C. Thoreen, D. Cheng, G. Marsischky, J. Roelofs, D. Finley, and S.P. Gygi. 2003. A proteomics approach to understanding protein ubiquitination. Nature biotechnology. 21:921-926.

Pickart, C.M., and R.E. Cohen. 2004. Proteasomes and their kin: proteases in the machine age. Nature reviews. Molecular cell biology. 5:177-187.

Ploger, R., J. Zhang, D. Bassett, R. Reeves, P. Hieter, M. Boguski, and F. Spencer. 2000. XREFdb: cross-referencing the genetics and genes of

69

mammals and model organisms. Nucleic acids research. 28:120-122.

Polier, S., Z. Dragovic, F.U. Hartl, and A. Bracher. 2008. Structural basis for the cooperation of Hsp70 and Hsp110 chaperones in protein folding. Cell. 133:1068-1079.

Polier, S., F.U. Hartl, and A. Bracher. 2010. Interaction of the Hsp110 molecular chaperones from S. cerevisiae with substrate protein. Journal of molecular biology. 401:696-707.

Prasad, R., S. Kawaguchi, and D.T.W. Ng. 2010. A Nucleus-based Quality Control Mechanism for Cytosolic Proteins. Molecular Biology of the Cell. 21:2117-2127.

Preker, P.J., K.S. Kim, and C. Guthrie. 2002. Expression of the essential mRNA export factor Yra1p is autoregulated by a splicing-dependent mechanism. RNA. 8:969-980.

Rampelt, H., J. Kirstein-Miles, N.B. Nillegoda, K. Chi, S.R. Scholz, R.I. Morimoto, and B. Bukau. 2012. Metazoan Hsp70 machines use Hsp110 to power protein disaggregation. EMBO Journal. 31:4221-4235.

Ravid, T., and M. Hochstrasser. 2008. Diversity of degradation signals in the ubiquitin-proteasome system. Nature reviews. Molecular cell biology. 9:679-689.

Raviol, H., H. Sadlish, F. Rodriguez, M.P. Mayer, and B. Bukau. 2006. Chaperone network in the yeast cytosol: Hsp110 is revealed as an Hsp70 nucleotide exchange factor. The EMBO journal. 25:2510-2518.

Reyes-Turcu, F.E., K.H. Ventii, and K.D. Wilkinson. 2009. Regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes. Annu Rev Biochem. 78:363-397.

Rio, D.C. 1993. Splicing of pre-mRNA: mechanism, regulation and role in development. Current Opinion in Genetics & Development. 3:574-584.

Rodriguez-Navarro, S., J.C. Igual, and J.E. Perez-Ortin. 2002. SRC1: an intron-containing yeast gene involved in sister chromatid segregation. Yeast. 19:43-54.

Rosenbaum, J.C., E.K. Fredrickson, M.L. Oeser, C.M. Garrett-Engele, M.N. Locke, L.A. Richardson, Z.W. Nelson, E.D. Hetrick, T.I. Milac, D.E. Gottschling, and R.G. Gardner. 2011. Disorder targets misorder in nuclear quality control degradation: a disordered ubiquitin ligase directly recognizes its misfolded substrates. Molecular cell. 41:93-106.

Ruby, S.W., and J. Abelson. 1991. Pre-mRNA splicing in yeast. Trends in Genetics. 7:79-85.

Rudiger, S., L. Germeroth, J. Schneider-Mergener, and B. Bukau. 1997. Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries. The EMBO journal. 16:1501-1507.

70

Russell, S.J., K.A. Steger, and S.A. Johnston. 1999. Subcellular localization, stoichiometry, and protein levels of 26 S proteasome subunits in yeast. The Journal of biological chemistry. 274:21943-21952.

Schlesinger, M.J. 1990. Heat shock proteins. The Journal of biological chemistry. 265:12111-12114.

Schmitt, A.P., and K. McEntee. 1996. Msn2p, a zinc finger DNA-binding protein, is the transcriptional activator of the multistress response in Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences of the United States of America. 93:5777-5782.

Schreiber, K., G. Csaba, M. Haslbeck, and R. Zimmer. 2015. Alternative Splicing in Next Generation Sequencing Data of Saccharomyces cerevisiae. PLoS One. 10:e0140487.

Schuermann, J.P., J. Jiang, J. Cuellar, O. Llorca, L. Wang, L.E. Gimenez, S. Jin, A.B. Taylor, B. Demeler, K.A. Morano, P.J. Hart, J.M. Valpuesta, E.M. Lafer, and R. Sousa. 2008. Structure of the Hsp110:Hsc70 nucleotide exchange machine. Molecular cell. 31:232-243.

Shaner, L., A. Trott, J.L. Goeckeler, J.L. Brodsky, and K.A. Morano. 2004. The function of the yeast molecular chaperone Sse1 is mechanistically distinct from the closely related hsp70 family. The Journal of biological chemistry. 279:21992-22001.

Shaner, L., H. Wegele, J. Buchner, and K.A. Morano. 2005. The yeast Hsp110 Sse1 functionally interacts with the Hsp70 chaperones Ssa and Ssb. The Journal of biological chemistry. 280:41262-41269.

Sherman, F. 2002. Getting started with yeast. Methods in enzymology. 350:3-41.

Shiber, A., W. Breuer, M. Brandeis, and T. Ravid. 2013. Ubiquitin conjugation triggers misfolded protein sequestration into quality control foci when Hsp70 chaperone levels are limiting. Molecular Biology of the Cell. 24:2076-2087.

Shirayama, M., K. Kawakami, Y. Matsui, K. Tanaka, and A. Toh-e. 1993. MSI3, a multicopy suppressor of mutants hyperactivated in the RAS-cAMP pathway, encodes a novel HSP70 protein of Saccharomyces cerevisiae. Molecular & general genetics : MGG. 240:323-332.

Shomura, Y., Z. Dragovic, H.-C. Chang, N. Tzvetkov, J.C. Young, J.L. Brodsky, V. Guerriero, F.U. Hartl, and A. Bracher. 2005. Regulation of Hsp70 Function by HspBP1: Structural Analysis Reveals an Alternate Mechanism for Hsp70 Nucleotide Exchange. Molecular cell. 17:367-379.

Shorter, J. 2011. The mammalian disaggregase machinery: Hsp110 synergizes with Hsp70 and Hsp40 to catalyze protein disaggregation and reactivation in a cell-free system. PLoS One. 6:e26319.

Sondermann, H., A.K. Ho, L.L. Listenberger, K. Siegers, I. Moarefi, S.R. Wente, F.-U. Hartl, and J.C. Young. 2002a. Prediction of Novel Bag-1 Homologs Based on Structure/Function Analysis Identifies

71

Snl1p as an Hsp70 Co-chaperone in Saccharomyces cerevisiae. Journal of Biological Chemistry. 277:33220-33227.

Sondermann, H., A.K. Ho, L.L. Listenberger, K. Siegers, I. Moarefi, S.R. Wente, F.U. Hartl, and J.C. Young. 2002b. Prediction of novel Bag-1 homologs based on structure/function analysis identifies Snl1p as an Hsp70 co-chaperone in Saccharomyces cerevisiae. The Journal of biological chemistry. 277:33220-33227.

Sondermann, H., C. Scheufler, C. Schneider, J. Höhfeld, F.-U. Hartl, and I. Moarefi. 2001. Structure of a Bag/Hsc70 Complex: Convergent Functional Evolution of Hsp70 Nucleotide Exchange Factors. Science. 291:1553-1557.

Sorger, P.K. 1991. Heat shock factor and the heat shock response. Cell. 65:363-366.

Sorger, P.K., and H. Pelham. 1987. Purification and characterization of a heat-shock element binding protein from yeast. The EMBO journal. 6:3035.

Sorger, P.K., and H.R. Pelham. 1988. Yeast heat shock factor is an essential DNA-binding protein that exhibits temperature-dependent phosphorylation. Cell. 54:855-864.

Spingola, M., L. Grate, D. Haussler, and M. Ares, Jr. 1999. Genome-wide bioinformatic and molecular analysis of introns in Saccharomyces cerevisiae. Rna. 5:221-234.

Stamm, S., S. Ben-Ari, I. Rafalska, Y. Tang, Z. Zhang, D. Toiber, T.A. Thanaraj, and H. Soreq. 2005. Function of alternative splicing. Gene. 344:1-20.

Stefani, M., and C.M. Dobson. 2003. Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution. Journal of molecular medicine. 81:678-699.

Summers, D.W., P.M. Douglas, C.H. Ramos, and D.M. Cyr. 2009. Polypeptide transfer from Hsp40 to Hsp70 molecular chaperones. Trends Biochem Sci. 34:230-233.

Summers, D.W., K.J. Wolfe, H.Y. Ren, and D.M. Cyr. 2013. The Type II Hsp40 Sis1 cooperates with Hsp70 and the E3 ligase Ubr1 to promote degradation of terminally misfolded cytosolic protein. PLoS One. 8:e52099.

Suzuki, K., and Y. Ohsumi. 2007. Molecular machinery of autophagosome formation in yeast, Saccharomyces cerevisiae. FEBS letters. 581:2156-2161.

Swaminathan, S., A.Y. Amerik, and M. Hochstrasser. 1999. The Doa4 deubiquitinating enzyme is required for ubiquitin homeostasis in yeast. Mol Biol Cell. 10:2583-2594.

Szabo, A., R. Korszun, F. Hartl, and J. Flanagan. 1996. A zinc finger-like domain of the molecular chaperone DnaJ is involved in binding to denatured protein substrates. The EMBO journal. 15:408.

Takayama, S., T. Sato, S. Krajewski, K. Kochel, S. Irie, J.A. Millan, and J.C. Reed. 1995. Cloning and functional analysis of BAG-1: a novel Bcl-2-binding protein with anti-cell death activity. Cell. 80:279-284.

72

Takeshige, K., M. Baba, S. Tsuboi, T. Noda, and Y. Ohsumi. 1992. Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction. The Journal of cell biology. 119:301-311.

Tamura, N., F. Lottspeich, W. Baumeister, and T. Tamura. 1998. The role of tricorn protease and its aminopeptidase-interacting factors in cellular protein degradation. Cell. 95:637-648.

Tanaka, K., T. Yoshimura, T. Tamura, T. Fujiwara, A. Kumatori, and A. Ichihara. 1990. Possible mechanism of nuclear translocation of proteasomes. FEBS letters. 271:41-46.

Thrower, J.S., L. Hoffman, M. Rechsteiner, and C.M. Pickart. 2000. Recognition of the polyubiquitin proteolytic signal. The EMBO journal. 19:94-102.

Tokuriki, N., and D.S. Tawfik. 2009. Chaperonin overexpression promotes genetic variation and enzyme evolution. Nature. 459:668-673.

Truman, A.W., K. Kristjansdottir, D. Wolfgeher, N. Hasin, S. Polier, H. Zhang, S. Perrett, C. Prodromou, G.W. Jones, and S.J. Kron. 2012. CDK-dependent Hsp70 Phosphorylation controls G1 cyclin abundance and cell-cycle progression. Cell. 151:1308-1318.

Tyedmers, J., A. Mogk, and B. Bukau. 2010. Cellular strategies for controlling protein aggregation. Nature reviews. Molecular cell biology. 11:777-788.

Udgaonkar, J.B. 2008. Multiple routes and structural heterogeneity in protein folding. Annual review of biophysics. 37:489-510.

Vabulas, R.M., S. Raychaudhuri, M. Hayer-Hartl, and F.U. Hartl. 2010. Protein folding in the cytoplasm and the heat shock response. Cold Spring Harb Perspect Biol. 2:a004390.

Varshavsky, A. 1997. The ubiquitin system. Trends in Biochemical Sciences. 22:383-387.

Verghese, J., J. Abrams, Y. Wang, and K.A. Morano. 2012. Biology of the Heat Shock Response and Protein Chaperones: Budding Yeast (Saccharomyces cerevisiae) as a Model System. Microbiology and Molecular Biology Reviews. 76:115-158.

Verghese, J., and K.A. Morano. 2012. A Lysine-Rich Region within Fungal BAG Domain-Containing Proteins Mediates a Novel Association with Ribosomes. Eukaryotic Cell. 11:1003-1011.

Vilardell, J., and J.R. Warner. 1994. Regulation of splicing at an intermediate step in the formation of the spliceosome. Genes & Development. 8:211-220.

Vogel, J.L., D.A. Parsell, and S. Lindquist. 1995. Heat-shock proteins Hsp104 and Hsp70 reactivate mRNA splicing after heat inactivation. Current biology : CB. 5:306-317.

Wall, D., M. Zylicz, and C. Georgopoulos. 1994. The NH2-terminal 108 amino acids of the Escherichia coli DnaJ protein stimulate the ATPase activity of DnaK and are sufficient for lambda replication. Journal of Biological Chemistry. 269:5446-5451.

73

Walter, S., and J. Buchner. 2002. Molecular chaperones--cellular machines for protein folding. Angewandte Chemie. 41:1098-1113.

Wegrzyn, R.D., and E. Deuerling. 2005. Molecular guardians for newborn proteins: ribosome-associated chaperones and their role in protein folding. Cellular and molecular life sciences : CMLS. 62:2727-2738.

Werner-Washburne, M., and E.A. Craig. 1989. Expression of members of the Saccharomyces cerevisiae hsp70 multigene family. Genome. 31:684-689.

Willmund, F., M. del Alamo, S. Pechmann, T. Chen, V. Albanèse, Eric B. Dammer, J. Peng, and J. Frydman. 2013. The Cotranslational Function of Ribosome-Associated Hsp70 in Eukaryotic Protein Homeostasis. Cell. 152:196-209.

Wolf, D.H., and W. Hilt. 2004. The proteasome: a proteolytic nanomachine of cell regulation and waste disposal. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 1695:19-31.

Xu, P., D.M. Duong, N.T. Seyfried, D. Cheng, Y. Xie, J. Robert, J. Rush, M. Hochstrasser, D. Finley, and J. Peng. 2009. Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation. Cell. 137:133-145.

Xu, X., E.B. Sarbeng, C. Vorvis, D.P. Kumar, L. Zhou, and Q. Liu. 2012. Unique peptide substrate binding properties of 110-kDa heat-shock protein (Hsp110) determine its distinct chaperone activity. The Journal of biological chemistry. 287:5661-5672.

Yaglom, J.A., A.L. Goldberg, D. Finley, and M.Y. Sherman. 1996. The molecular chaperone Ydj1 is required for the p34CDC28-dependent phosphorylation of the cyclin Cln3 that signals its degradation. Molecular and Cellular Biology. 16:3679-3684.

Yamamoto, A., Y. Mizukami, and H. Sakurai. 2005. Identification of a novel class of target genes and a novel type of binding sequence of heat shock transcription factor in Saccharomyces cerevisiae. The Journal of biological chemistry. 280:11911-11919.

Yassour, M., T. Kaplan, H.B. Fraser, J.Z. Levin, J. Pfiffner, X. Adiconis, G. Schroth, S. Luo, I. Khrebtukova, A. Gnirke, C. Nusbaum, D.A. Thompson, N. Friedman, and A. Regev. 2009. Ab initio construction of a eukaryotic transcriptome by massively parallel mRNA sequencing. Proceedings of the National Academy of Sciences of the United States of America. 106:3264-3269.

Yost, H.J., and S. Lindquist. 1991. Heat shock proteins affect RNA processing during the heat shock response of Saccharomyces cerevisiae. Molecular and Cellular Biology. 11:1062-1068.

Regulation of Hsp70 function by nucleotide-exchange factors919735/FULLTEXT01.pdfRegulation of Hsp70...

Documents

Transcript of Regulation of Hsp70 function by nucleotide-exchange factors919735/FULLTEXT01.pdfRegulation of Hsp70...