Proteins targeted to the endoplasmic reticulum can end up in the ER, golgi (trans, median, cis) Plasma membrane, secretedor in lysosomes

Studies with proteins found in the ER would indicate that these proteins remain in the ER. This means that they are notpassed to the golgi or plasma membrane.

Cloning a Protein Receptor geneAnalyis of a large number of proteins found in the ERfrom yeast and animal systems identified the amino acid sequence KDEL in the carboxy region of these proteins

It was proposed that a protein in the ER compartmentbound these proteins and in some manner kept then in the ER location - It was proposed that a KDEL receptor existed. However there was no information as to the identity of this receptor.

5’ CAPAAA 3’SRPCYCLRIBOSOME CYCLEER MEMBRANEERLUMENCYTOSOL= SIGNAL RECOGNITION PARTICLE(SRP)= SRP RECEPTOR= RIBOSOME RECEPTOR

Design library screen to identify receptorYeast - is a useful micro-organism in that it is Eukaryoticand yet can be manipulated easily. Especially it cangrow on a variety of media and easily mutagenised.

Yeast can normally grow on sucrose as a sole carbon source, it does this by secreting an enzyme called invertase which breaks sucrose down into glucose and fructose. These monosaccharides are then taken up by yeast and used.

Invertase+

Inactivate invertase gene in yeastso it cannot grow on sucrose. It should be noted that it can be maintained on glucose etc. genes can be inactivated by various means and such strains are available commerically

SucroseInvertase-GlucoseInvertase-

Invertase-Invertase -KDEL+A new invertase gene is put into the mutant yeast strain. However this invertase genewill have a KDEL sequence in the carboxy terminus and so will not be secreted from the cell. It will be kept in the ER. Therefore the yeastcells will not grow on sucrose.

SucroseInvertase-Invertase -KDEL+Glucose

The yeast strain(Invertase- , Invertase-KDEL+)is mutagenised randomlyIt will be found that a smallnumber of colonies will growon sucrose. The reason is that when the yeast strainwas mutagenised, the KDEL receptor gene will be mutatedat a low frequency, all gene willbe. When the KDEL receptor geneis mutated the invertase is not retained in the ER, it is secreted and so the yeast can growon sucrose. We now have a mutation for the KDEL receptor.

SucroseInvertase-Invertase -KDEL+KDEL-Receptor-Glucose

SucroseTo identify the gene for the KDELreceptor, the yeast strain growing on sucrose is complemented with a cDNA library from wild-type yeast.This will complement the mutation in the KDEL receptor gene.

Invertase -KDEL+KDEL-Receptor-GlucoseThe colony that receives the functioning KDEL gene will not grow as the invertase gene will be retained in the ER. To identifythis colony the transformed yeast are grown on glucose and replica platedonto sucrose. The colony that fails to grow on sucrose has the KDEL gene. This colony is grown on glucose and the gene isolated

How do we test function of yeast homologues in animals and plants as lethal if mutated or deleated - in both yeast and higher eukaryotes

NCCNOutermembraneInnermembraneTim17Tim23

Tim17:23 Membrane location (Yeast)

AOX AOX

Tim17:23

•Import channel on inner membrane Recently shown that channel activity associated with Tim23. Four membrane spanning section has voltage dependant channel activity and the N-terminal region which binds presequences gives channel specificity.

•Role in Apoptosis or programmed cell death ?

H.s TIM17S.c TIM17At TIM17-2

0 50 100 150 200 250

At TIM17-5

0 40 80 120 160 0 40 80 120 0 40 80 120 160

AT-2AT-5S.cH.s

AT-2AT-5S.cH.s

AT-2AT-5S.cH.s

AT-2AT-5S.cH.s

AT-2AT-5S.cH.s

TIM17

1 50MGTPETSREP CPDRILDDIG GAFGMGAVGG SAFHFIKGTY NSPKGSRFVG

51 100GTQSVSMNAP RTGGSFAVWG GLFSTFDCTM VYLRQKEDPW NSIIAGAATG

101 150GFLSMRQGAG AASRSTIFGG VLLALIEGAG IMLNKVLAQP QNMMMEDPGM

151 200QGMPGMQGMQ GMPGMPGMQG MPGMQGMQMG QMQSQAQIRS ESQNQNTASS

201 243SSSSSWFGGL FDKKKEEVHP GSESKTEVLE SFDAPPVPSF EFK

Arabidopsis Tim17

TIM23

S.c TIM23 H.s TIM23

0 40 80 120 160 200 0 50 100 150 200 250 0 50 100 150 200 250

At TIM23

1 50Yeasttim23 MSWLFGDKTP TDDANAAVGG QDTTKPKELS LKQSLGFEPN INNIISGP.G Arabtim23 .......... .......... .... .. ...........MA ANNRSDHGSD

51 100Yeasttim23 GMHVDTARLH PLAGLDKGVE YLDLEEEQLS SLEGSQGLIP SRGWTDDLCY Arabtim23 ENTRLYNPYQ NYEVPINKSQ YLYKLPTSPE FLFTEEALRQ RRSWGENLTF

101 150Yeasttim23 GTGAVYLLGL GIGGFSGMMQ GLQNIPPNSP GKLQLNTVLN HITKRGPFLG Arabtim23 YTGTAYLGGS VAGASVGVIT GVKSFESGDT TKLKINRILN SSGQTGRTWG

151 200Yeasttim23 NNAGILALSY NIINSTIDAL RGKHDTAGSI GAGALTGALF KSSKGLKPMG Arabtim23 NRIGIIGLVY AGIESGIVAA TDRDDVWTSV VAGLGTGAVC RAARGVRSAA

201 227Yeasttim23 YSSAMVA.AA CAVWCS..VK KRLLEK.Arabtim23 VAGALGGLAA GAVVAGKQIV KRYVPI*

Arabidopsis Tim23

Outermembrane

AOX AOX

Characterise Tim17:23 in Plants

Q - Given the differences observed does it play the same role in plants via the same mechanisms as elucidated in yeast?

The acquisition of mitochondria(or endosymbiosis) hasbeen proposed to be vital for the evolution of multi-cellular organisms. Yeast is an excellant model system but cannot claim to represent diverse multicellular organisms that display developmental and tissue specific functions.

AOX AOX

Function of AtTim17 and AtTim23

Knock-out mutants of yTim17 and yTim23 are lethal so must use a replacement strategy to see if plant genes can complement yeast genes.

Yeast 2N+XNormal+X+Yeast 2NExtra Functional copyof gene on plasmidNormal+X++Yeast 2NExtra Functional copyof gene on plasmidPlant Gene on PlasmidNormalTetradAnalysis++++++X++Yeast NYeast NYeast NNormalNormalNormalX++Yeast NNormal

++++++X++X++Yeast NNormalYeast NNormalYeast NNormalYeast NNormalCure Cells of Plasmid

Carrying Yeast Gene

++++X+X+Yeast NNormalYeast NNormalYeast NGrowth will depend on ability of Plant geneto complementYeast NGrowth will depend on ability of Plant geneto complement

EUROPEAN  ..........SACCHAROMYCES  ....................CEREVISIAE  ..............................ARCHIVE FOR  ........................................FUNCTIONAL ANALYSIS

Yeast deletions for 5943 genesHaploid non essential genesDiploid essential genes

Deletion strains for mitochondrial importTim 10, 11, 12, 17, 22, 23, 44, 54Tom 5, 6, 7, 20, 22, 72, 34, 37, 40, 71, 72

Deletions have kanMX4 inserted into gene of interest

tetrad

kanMX4

kanMX4

Tim17

Tim17

kanMX4 kanMX4Tim17

Tim17

2:2 genotype segregation

Tetrad analysis

1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10ABCD

ABCD

ABCD

ABCD

WA-ura

YPGc + geneticin

WA-meth

WA-lys

Tim17 Tim23

ΔTim 17 ΔTim23yTim17AtTim17-2AtTim17-5AtTim17-2Stop 143+------+yTim23AtTim23--+-

AOX AOX

Function of AtTim17 and AtTim23

•Different AtTim17 genes have different activities

•C-terminal region of AtTim17-2 prevents it activity in yeast which implies a different function in plants

•AtTim23 cannot complement a yeast mutant implying different functions