I'e'oq The mammalian mitochondrial Hsp70 chaperone system, new GrpE-like members and novel organellar substrates Submitted by Dean Jason Naylor BSc. (Hons.) A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy The Faculty of Agricultural and Natural Resource Sciences The University of Adelaide, V/aite Campus, Department of Horticulture, Viticulture and Oenology Glen Osmond, South Australia 5064, Australia August, 1999 Table of contents , Table of contents Table of contents List of ab brev iatíons........ Summary....,. State ntent of authors lüp Acknowledgments xt List of publications xtL CHAPTER I General introduction 1..1. Scope of chaperone existence and focus of this review.......... .............................1 1.2 The discovery of heat shock (stress) proteins and molecular chaperones................. ............3 1.4 The molecular chaperone concept........ ................. 11 1.5 The Hsp70 molecular chaperone system......... ........12 1.5.1 E. coli constitutes a model systemfor the study of eukaryotic molecular chaperones...................12 1.5.2 The E. coli DnaK (Hsp70) chaperone systenx.. I3 1.5.3 Structure-function relationship of the DnaK(Hsp70)/DnaJ(Hsp40)/GrpE system. .t6 1.5.3.1 DnaK (Hsp70) component.. t6 1.5.3.2 DnaJ (Hsp40) component. 1.5.3. 3 GrpE component, 21 1.5.4 Reaction cycle of the DnaK (Hsp7))/DnaJ (Hsp4})/GrpE system. 22 1.5.5 In some Hsp70 systems, GrpE may be replaced by additional co-factors that broaden the functions of Hsp70 chaperones. ......................24 1.5.6 The DnaK (Hsp70) substrate binding motif.. 26 1.5.7 The contribution of molecular chaperones to the folding of newly synthesised proteins and those unfolded by stress.. L5.8 The cooperation of molecular chaperone systems during de novo protein foldíng and the reþlding of stress-denatured proteins. 28 1.6 The components involved in mitochondrial protein import, folding and assemb1y.................................30 1.6.1 The translocation of preproteins across the mitochondrial inner membrane requires the matrix located mt-Hsp70.... 3I 1.6.2 The requirement of a distinct mt-Hsp7} systemÍor protein (re)þlding and assemb|y................ JJ 1.6.3 Protein import and folding in mammaliqn mitochondria.. 36 1.7 Roles of chaperones in protein degradation within E. coli and mitochondria........... 1.7. I M any stress proteins are proteas es...,............,. I .7.2 Protein degradation within the mitochondrion. L7.3 Chaperone assisted proteases of E. coli...... 1.7.3.1 The kt (Lon) protease, 1.7. 3.2 The Clp proteases............ 1.7. 3. 3 The FtsH ( HflB ) proteas e............. Table of contents ,l 1.7.4 Chaperone assisted proteases within the mitochondrion.....'..'..'.'....' ..'.."................41 1.7.4.1 Homologues of the La (Lon) protease..............'... ...'.....'....'41 L7.4.2 Homologues of the Clp family and ClpP proteolytic subunit..'. '....".'..'.. 4I 1.7.4.3 Homologues of the FtsH (HfIB) protease................. .'.........42 CHAPTER 2 Affinity-purification and identification of a mammalian mito chondrial GrpE-Iike protein (mt-GrpØ# I ) 2.2 MATERIALS AND METHODS ......,..46 2.2.1 Preparation of a DnaK-affinity column.. 46 2.2.2 I s olqt ion of s olub le mito c hondrial proteins................ .46 2.2.4 Protein and peptide sequence analysis. 47 47 ,a 2.2.6 Coomassie Brilliant Blue staining of proteins after SDS-P{GE................. 2.3 RESULTS AND DISCUSSION................... 48 CHAPTER 3 Isolation and choracterisation of a cDNA encoding mammalian mt-GrpE#l 3.1 INTRODUCTION 51 3.2 MATERIALS AND METHODS ........,52 3.2. I Synthesis of oli gonucleo tide primers........ 52 3.2.2 Isolation of nRNA, RT-PCR, cDNA library screening and 5'-RACE analysis, .52 3.2.3 Production of anti-mt-GrpE#1 and antïHsp60 polyclonal antibodies....... 53 3.2.4 Western blot analysis 53 3.2.5 Northern analysis. 54 3.2.6 Growth of tissue culture ce\\s...,.....,.... .54 3.2.7 Stress treatments, metabolic labelling andfractionation of tissue culture ce\\s.............. 54 3.2. 8 Semi-quantitative RT-P CR. 55 3.3.1 Cloning of rat mt-GrpÛ#l cDNA. 56 3.3.2 Members of the GrpEfamily exhibit a relatively low degree of sequence identity at the amino acid level 3.3.3 mt-GrpE#l is a low abundqnce mitochondrial protein of ub i quito us ap p earanc e in mamma lian or 7ans....... 58 3.3.4 mt-GrpE#I synthesis is induced slightly by amino acid analogue treatment but not by heat shock.. 59 Table of contents ItL CHAPTER 4 Definition of two functional mitochondrial GrpE-like proteins in mammalian cells 4.2 MATERIALS AND METHODS .........63 4.2. I I mmunolo g ical te c hniq ue s. ,63 4.2.2 Nucleotide sequence analysis........ 63 4.2.3 Southern blot analysis........ 63 4.2.4 Expression of mouse mt-GrpØ#2, rat mt-GrpE#l and rat mt-Hsp7} in E. coli.. 64 4.2.5 Native P4G8........... 66 4.2.6 ATPase assay......... 66 4.2.7 RNA dot blot ana|ysis............... 66 4.3 RESULTS ANDDISCUSSION.................. 67 4. 3. I Ev idenc e for the existenc e of a distinct cyto s olic GrpE- like protein. 67 4.3.2 ldentification of a second mammalian mt-GrpE cDNA.. 68 4.3.3 mt-GrpE#2 is present in mitochondria. 69 4.3.4 Properties of mt-GrpE#l and #2 and interaction with DnaK...,....... 70 4.3.5 mt-GrpE#I qnd #2 are functional co-chaperones for mt-Hsp7}..... '7 1 4. 3.6 Expression of mt-GrpE#2.............. 72 CHAPTER 5 Characterisation of several Hsp70 interacting proteins from mammalian or I anelle s 74 5.2 MATERIALS AND METHODS 76 5.2. 1 D naK- and ATP -ffinity c hromato çraphy.......... 76 5.2.2 Protein s equence analysis........ 76 78 81 CHAPTER 6 Mitochondria and peroxisomes may contain distinct isoforms of 2,4-dienoyl CoA reductase 85 6.2 MATERIALS AND METHODS 88 6. 2. I P ur ificatio n of the 2,4- di e noy I- C oA r e duc t a s e s.... 8B 6.2.2 Synthesis of oligonucleotide primers. 88 6.2.3 Southern blot anaLysis........ .89 6.2.4 Isolation of mRNA, RT-PCR, cDNA library screening and 5'-RACE analysis..... ,89 6.2. 5 Immunological techniques.... 89 6.2.6 Expression of rat px-2,4-DCR#1 in E. co\i...... 90 6.2.7 Enzyme assays. .91 Table of contents iv 6.2. 8 Northern blot analysis........ 91 ot 6. 3. 1 DnaK-affiníty purffication of rat px-2,4-D CR# I and mt-2,4- D CR# I 6.3.2 Cloning of rat px-2,4-DCR#I cDNA.. 93 6.3.3 Rat px-2,4-DCR#I exhibits significant sequence identity with members of the 2,4-dienoyl CoA reductase family at the amino acid \evel............ .................... 93 6. 3. 4 Chqracteris atíon of re c ombinant rat px-2,4 - D C R# 1 ...,.,... 94 6. 3. 5 Expression of px-2,4-D CR# I and mt-2,4- DCR# I tans uipts. ....'.'...'..',..,,'.,,,96 CHAPTER 7 Summary andfuture directions 101 REFERENCES. 104 Abbreviations v List of øbbreviations A. absorbance adenosine 5' -diphosphate adenosine 5' -triphosphate A2c........... L-azetidine-2-carboxylic acid BAG-1...... Bcl-2-associated anti-death gene I Bcl-2......... B cell lymphoma 2 BiP immuno globulin-binding protein N,N' -methylene-bis-acrylamide Bos taurus bovine serum albumin base pair(s) chaperonin containing TCP-1 carboxyl-terminus complementary DNA C aeno rhabditis ele g ans acetonitrile coenzyme A control counts per minute cpn. chaperonin CyP........... cyclophilin Da............. Dalton DCR......... dienoyl CoA reductase DHFR....... dihydrofolate reductase D. melanogaster ... Drosophila melanogaster DMEM..... Dulbecco's modified Eagle medium DNA......... deoxyribonucleic acid DTT.......... 1,4-dithiothreitol EB equilibration buffer ECL.......... enhanced chemiluminescence E. coli Escherichia coli Abbreviations vi EDTA... ...... ethylenediaminetetracetic acid ER........ ,..... endoplasmic reticulum ESI-MS electrospray ionisation mass spectrometry EST...... .... expressed sequence tag GimC... ..... genes involved in microtubule biogenesis complex Glycine ..... aminoacetic acid h........... .... hour(s) Hap...... .... Hsp70/Hsc70-associating protein HEPES..... N-[2-Hydroxyethyl]piperazine-N'-|2-ethanesulfonicacidl Hip............ Hsc70-interacting protein Hop........... Hsc70ÆIsp9O-organising protein HPLC....... high pressure liquid chromatography Hs............. heat shock Hsp........... heat shock protein Hsc70....... constitutive (or cognate) isoform of Hsp70 Hsp70....... 70 kDa heat shock protein HRP.......... horseradish peroxidase H. sapiens Homo sapiens IMAC....... immobilisedmetalaffinitychromatography IPTG......... isopropyl-1-thio-B-D-galactopyranoside LB Luria-Bertani mrn................. minute MFE............... ........ multifunctional enzyme M. musculus Mus musculus Mops. 3 - [N-Morpholino]propanesulfonic acid MPP......, ........ mitochondrial processing peptidase mRNA... ,........ messenger RNA MSF......, mitochondrial import stimulation factor mt.......... ........ mitochondrial mtTFA...... mitochondrial transcription factor A NAC..... nascent polypeptide-associated complex NADPH p-nicotinamide adenine dinucleotide, reduced form NMR..... nuclear magnetic resonance Abbreviations vií N-terminus amino-terminus ORF.......... open reading frame PAGE....... polyacrylamide gel electrophoresis PBS.......... phosphate buffered saline PCR.,........ polymerase chain reaction PBF........... presequence binding factor pI.............. isoelectric point PMSF....... phenylmethylsulphonyl fluoride Ponceau S............... 3-hydroxy-4-[2-sulfo-4-(sulfophenylazo) phenylazo]-2,7- naphthalenedisulfonic acid PPIase....... peptidyl-proyl cisltrans isomerase PTS-1....... peroxisomal targeting sequence type-1 px.............. peroxisomal R4CE....... rapid amplification of