The Conserved Carboxyl Terminus and Zinc Finger-like Domain of the Co-chaperone Ydj1 Assist Hsp70 in Protein Folding

• DOI: 10.1074/jbc.273.10.5970
• Publication Date: 2002-07-26T15:13:03Z
• Authors: Zhen Lu; Douglas M. Cyr
• Keywords: Chaperone (clinical); C-terminus; Foldase

Ydj1 is a member of the Hsp40 (DnaJ-related) chaperone family that facilitates cellular protein folding by regulating Hsp70 ATPase activity and binding unfolded polypeptides. contains four conserved subdomains appear to represent functional units. To define action these regions, protease-resistant fragments mutants were analyzed for activities exhibited unmodified protein. The mutant proteins unable support growth yeast at elevated temperatures found have alterations in J-domain (Ydj1 H34Q), zinc finger-like region C159T), carboxyl terminus G315D). Fragment (1–90) small portion G/F-rich could regulate but not suppress aggregation model rhodanese. H34Q bind functions independently was sufficient activity. (179–384) rhodanese Hsp70. DnaJ missing J-domain, region, major region. G315D severe defects its ability form complexes with luciferase. appeared participate C159T stable inefficient refolding denatured function conjunction fold proteins. However, does require an intact These data suggest combined are required cooperate facilitate cell. make up essential molecular chaperones specify processes facilitated (1Cyr D.M. Langer T. Douglas M.G. Trends Biochem. Sci. 1994; 19: 176-181Abstract Full Text PDF PubMed Scopus (402) Google Scholar, 2Caplan A.J. Cyr Mol. Biol. Cell. 1993; 4: 555-563Crossref (194) 3Georgopoulos C. 1992; 17: 295-299Abstract (201) 4Silver P.A. Way J.C. 74: 5-6Abstract (189) 5Hartl F.U. Nature. 1996; 381: 571-579Crossref (3130) Scholar). Prokaryotic eukaryotic cells contain multiple act specific pairs different locations via mechanisms (6Brodsky J.L. Hamamoto S. Feldheim D. Schekman R. J. Cell 120: 95-102Crossref (130) 7Cyr Chem. 269: 9798-9804Abstract 8Schlenstedt G. Harris Risse B. Lill Silver 1995; 129(4): 979-988Crossref (134) 9Cyr Gething M.-J. Guidebook Molecular Chaperones Protein Folding Factors. Oxford University Press, Oxford, United Kingdom1997: 89-95Google Escherichia coliDnaJ, progenitor family, promote folding, degradation misfolded proteins, bacteriophage DNA replication (3Georgopoulos 10Zylicz M. Philos. Trans. Soc. Lond. B 339: 271-278Crossref (46) 11Sherman Goldberg A.L. EMBO 11: 71-77Crossref (173) mammalian Auxillin directs uncoat clathrin-coated vesicles (12Barouch W. Prasad K. Greene L. Eisenberg E. Biochemistry. 1997; 36: 4303-4308Crossref (58) 13Jiang R.-F. Greener Barouch 272: 6141-6145Abstract (64) cysteine string Drosophilia palmitolyated localized neurosecretory vesicles, where it proposed direct catalyze putative assembly/disassembly events neurotransmitter release (14Braun J.E.A. Wilbanks S.M. Scheller R.H. 271: 25989-25993Abstract (124) 15Chamberlain L.H. Burgoyne R.D. 322: 853-858Crossref (106) Tim44 component import machinery mitochondrial cognate drive translocation into matrix space (16Ungermann Neupert Science. 266: 1250-1253Crossref (225) 17Ungermann Guiard 15: 735-744Crossref (90) 18Cyr Bioenerg. Biomembr. 29: 29-34Crossref (7) 19Lill Nargang F.E. Curr. Opin. 8: 505-512Crossref (65) 20Schatz 31763-31766Abstract (227) cytosolic deliver preproteins organelle membranes (21Atencio D.P. Yaffe M.P. 12: 283-291Crossref (122) 22Caplan 71: 1143-1155Abstract (219) Scholar), refold substrates (23Caplan Langley Wilson E.M. Vidal 270: 5251-5257Abstract (107) 24Kimura Y. Yahara I. Lindquist 268: 1362-1365Crossref ubiquitin-dependent (25Lee D.H. Sherman M.Y. 16: 4773-4781Crossref (125) polypeptides ATP-dependent reaction cycle, ADP-form exhibiting higher affinity than ATP-form (26Palleros D.R. Welch W.J. Fink Proc. Natl. Acad. U. A. 1991; 88: 5719-5723Crossref (281) It helps stabilize Hsp70-polypeptide through promoting conversion Hsp70-ATP Hsp70-ADP 27Liberek Marszalek Ang Georgopoulos Zylicz 2874-2878Crossref (693) 28Langer Lu Echols H. Flanagan Hayer M.K. Hartl 356: 683-689Crossref (793) 29McCarty J.S. Buchberger Reinstein Bukau 249: 126-137Crossref (349) some instances, can bound (7Cyr subset members also recognize aspects non-native structure intermediates manner subsequent interactions between them (28Langer 30Wickner Hoskins McKenney 7903-7907Crossref (138) 31Cyr FEBS Lett. 359: 129-132Crossref (115) 32Wawrzynow 19300-19306Abstract (102) 33Minami Hohfeld Ohtsuka F.-U. 19617-19624Abstract (282) In absence Hsp40, complex formation be inefficient, which leads substrate 34Wagner Arlt van Dyck 13: 5135-5145Crossref (208) Sequence analysis suggests regulatory actions catalyzed sequence motifs originate from regions termed domain, corresponds amino-terminal 70 amino acid residues all responsible regulation ATP hydrolytic cycle NMR demonstrates α-helical conformation, helices I II lying anti-parallel orientation III IV (35Hill R.B. J.M. Prestegard J.H. 34: 5587-5596Crossref (69) 36Qian Y.Q. Patel McColl D.J. 260: 224-235Crossref (139) 37Szyperski Pellecchia Wall Wuthrich 91: 11343-11347Crossref (141) dictated loop formed HPD tripeptide members. Mutations motif faces abrogate interact (38Feldheim Rothblatt 3288-3296Crossref 39Tsai 9347-9354Abstract (212) 40Wall 5446-5451Abstract 41Dey Caplan Boschelli F. 7: 91-100Crossref Functions clear. thought as flexible spacer other (4Silver case DnaJ, order productive coli occur (42Karzai A.W. McMacken 11236-11246Abstract (202) 43Wall 2139-2144Abstract (113) because several lack still Hsp70, this (13Jiang 14Braun Cysteine-rich repeats resemble C4 domains comprise domain play role polypeptide (44Banecki Liberek Wawrzynow Bertoli Tanfani 14840-14848Abstract (137) 45Szabo Korszun 408-417Crossref (276) Fragments capable suppressing (45Szabo involved lies adjacent typically present has been determined. further mechanism metabolism, we carried out structure-function Ydj1. identified activities. independent plays observed substrates. demonstrate units coordinated assist Ssa1 protein, remainder text, overexpressed strain MW141 purified ATP-agarose, DEAE, hydroxyapatite chromatography established techniques (46Cyr X. 267: 20927-20931Abstract open reading frames Ydj1, (39Tsai (24Kimura Scholar) subcloned pET9d, coli, ion-exchange described previously tagged six histidine metal chelate experiments C159T, histidine-tagged version utilized control. both behaved similarly on columns, ran dimers gel filtration columns (data shown). Therefore, forms grossly misfolded. Histidine-tagged temperature sensitive phenotype ΔYdj1 strains same efficiency Additionally, His-Ydj1 identically nontagged assays below His-tagged purification steps dimer columns. (0.5 mg/ml) digested trypsin or proteinase K (PK) 1The abbreviations used are: PK, K; DTT, dithiothreitol; PAGE, polyacrylamide electrophoresis. concentrations indicated digestion buffer (150 mm KCl, 25 Hepes, pH 7.4, 10 DTT) 1 h °C. Digestions terminated addition phenylmethylsulfonyl fluoride final concentration mm. Protease-resistant liberated then SDS-PAGE 12.5 16% acrylamide gels PK digestions, respectively. Digested (4.0 μg) run resolve products proteolytic each other. Gels soaked 20 min transfer (10 mm3-cyclohexylamino-1-propanesulfonic made 10% methanol). electrophoretically transferred polyvinylidene Bio-Rad Mini-Wet cell blotter 60 power supply set 50 V constant voltage. Membranes water 5 remove stained Coomassie Blue R-250 illuminate positions fragments. excised membranes, identity first determined Perkin-Elmer Applied Biosystems Microsequencing Apparatus. weights limited proteolysis Perseptive (Framingham, MA) Voyager Elite MALDI-TOF mass spectrometer. Aliquots material (5 μl) mixed saturated solution α-cyano-4-hydroxy-cinnamic water:acetonitrile (50:50) mixture acidified 0.1% trifluoroacetic acid. A 1-μl aliquot spotted onto gold plate target. Ionization sample nitrogen laser operating 337 nm. delayed extraction method determination weight. Measurement ion flight times drift spectrometer Tektronix (Beaverton, OR) TDS784A oscilloscope. instrument calibrated external weight standards. analyze (1–383), 2 μg/ml 30 inactivated 0.5 mmphenylmethylsulfonyl fluoride, (1–383) analyzed. At concentration, almost completely converted less 1% contamination (Fig. B, lane 3). levels contaminants preparation calculated insufficient account (1–383). generate (102–394), (179–384), analysis, digestions two out. Then, generated separated high performance liquid chromatography, concentrated, immediately assayed (102–394) virtually free (1–90), mg (1 1–2.0 45 adjusted so efficiently only minimal (see Fig. C, separate protease mixtures loaded Bio-Select G-250 column. column developed mobile phase consisting 150 mmHepes, DTT pumped 0.6 ml/min back pressure 850 psi. Fractions ml) collected Biologic medium system identify peaks. eluted 7.8 ml, 9.0 ml. recovery step 40% starting material. Gel resolved majority 3% fragment remained, 5% contaminant. quantity preparations assays. prepare (179–394) under conditions listed above, 7.5 PK. representative 5. unidentified lower generated, samples treated above. 8.0 ml phase, fraction routinely 93% pure 4% products. level contaminating bands attributed (179–384). larger fragments, 2.0 3) peak about 95% contained broad band gels, microsequence had corresponded 1–6 D) Analysis peptides spectroscopy 9596 Da size (1–90). We contaminate too low fraction. Purified incubated containing 80 mmMgCl2, μm ([α-32P]ATP, 2.0–3.0 × 105 cpm/pmol) Reactions placed ice, duplicate 2-μl aliquots ADP thin layer PEI cellulose plates Spontaneous subtracted prior calculations rates hydrolysis. kinetics linear least experimental Rates light scattering (31Cyr Briefly, bovine (50 μm; Sigma) °C 6 m guanidinium-HCl buffered fresh DTT. Denatured diluted 100-fold 300 μl composed mmKCl, When present, respective added monitoring increases over time spectrophotometer 320 nm Refolding firefly luciferase (Promega) (47Levy E.J. McCarty Chirico 368: 435-440Crossref (68) Luciferase (13 42-fold denaturation (25 MgCl2, guanidinum-HCl, DTT). allowed proceed 40 °C, removed 125 ATP) assay reagent (Promega). measured Turner TD-20/20 luminometer. (1.6 μm), (0.8 μm) reactions when 17-fold omitted, consistent previous reports Firefly (7.5 100 stock mguanidinium-HCl, mmDTT 292.5 give 2.5 μm. Where indicated, After 20-min incubation period allow form, Novarose SE-1000/17 (Bio-Rad) equilibrated flow rate ml/min. determine migrated, fractions collected, 400 precipitated 1.2 acetone. Pelleted resuspended gels. quantitated densitometry. luciferase, phase. eluted, whereas 50% native recovered (2.5 injected column, no alteration migration either observed. lucif