J-domain proteins promote client relay from Hsp70 during tail-anchored membrane protein targeting
نویسندگان
چکیده
J-domain proteins (JDPs) play essential roles in Hsp70 function by assisting client trapping and regulating the ATPase cycle. Here, we report that JDPs can further enhance targeting competence of Hsp70-bound during tail-anchored protein (TA) biogenesis. In guided-entry-of-tail–anchored pathway yeast, nascent TAs are captured cytosolic sequentially relayed to downstream chaperones, Sgt2 Get3, for delivery ER. We found two JDPs, Ydj1 Sis1, parallel support TA ER vivo. Biochemical analyses showed that, while Sis1 differ their ability assist trapping, both transfer Sgt2. The regulate cycle is enhancing vitro supporting insertion These results demonstrate a role conformation clients membrane family comprises central hub chaperone network maintains cellular homeostasis functions every stage life cycle, from de novo folding, transport, aggregate remodeling degradation (1Clerico E.M. Tilitsky J.M. Meng W. Gierasch L.M. 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Goloubinoff Huerta-Cepas Kirstein Liberek K. Nagata Nillegoda N.B. et al.Function, evolution, proteins.Cell Stress Chaperones. 2019; 24: 7-15Crossref 18Sahi Network general specialty cytosol.Proc. Natl. Acad. U. 2007; 104: 7163-7168Crossref (115) Class such Ydj1, N-terminal J-domain, glycine/phenylalanine-rich linker, CTDI/CTDII sites, dimerization domain. B represented share same organization except they lack CTD-I JDPs. C specialized involved translation, pre-mRNA splicing, clathrin-binding (19Krantz K.C. Puchalla Thapa Kobayashi Bisher Viehweg Carr C.M. Rye H.S. Clathrin coat disassembly Hsc70/Ssa1p auxilin/Swa2p observed single-particle burst analysis spectroscopy.J. 2013; 288: 26721-26730Abstract (17) 20Sahi T. Inada Pleiss J.A. Cwc23, critical splicing dispensable domain.Mol. 30: 33-42Crossref 21Weyer F.A. Gumiero Gesé G.V. Lapouge Sinning I. 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Macek Endo Kalbacher Rapaport Cytosolic biogenesis proteins.J. 217: 3091-3108Crossref (43) bind short cyclic β-hairpin newly synthesized Tom40, suggesting it plays direct recognition (25Jores Two Apj1 (Class A) Jjj3 C), were glycosylphosphatidylinositol-anchored (26Ast Cohen Schuldiner targets SRP-independent endoplasmic reticulum.Cell. 152: 1134-1145Abstract (121) Finally, Xdj1 Djp1 B) efficient assembly translocase outer (27Opaliński Ł. Priesnitz Wenz L.-S. Oeljeklaus Warscheid Becker Recruitment J-proteins TOM receptors promotes biogenesis.Cell Rep. 2036-2043.e5Abstract outer-membrane Mim1 (28Papić Elbaz-Alon Koerdt S.N. Leopold Worm Jung demonstrates specificity between cochaperone protein.Mol. 33: 4083-4094Crossref (53) Scholar), respectively. Despite abundance vivo data, organellar remains unclear. question guided entry (GET) pathway, delivers (TAs) harboring transmembrane (TMDs) highly organisms. single TMD near terminus processes organelle membranes, vesicular trafficking, quality control, apoptosis (29Chio U.S. Cho Shan S.-O. Mechanisms insertion.Annu. Dev. 417-438Crossref (54) 30Hegde R.S. Keenan R.J. Tail-anchored reticulum.Nat. 12: 787-798Crossref (197) Recently, demonstrated released ribosome Ssa1, protects TMDs aggregation crucial maintaining translocation-competent (31Cho relay Hsp70-cochaperone cascade safeguards targeting.EMBO 37e99264Crossref Ssa1 initiates energetically downhill events, first then factor Get3 Get1/2 receptor complex membrane. GET established how cochaperones unknown. work, show representing redundantly GET-dependent Unexpectedly, biochemical assists preventing aggregation, does not. On other hand, subsequent was Ssa1-bound Our uncover new competence. asked TAs. Because α precursor hypothesized also To test hypothesis, used set isogenic strains expression YDJ1, SIS1, genes under control tetracycline-repressible promoter (tet-YDJ1, tet-SIS1, tet-YDJ1/tet-SIS1; Scholar)). depleted and/or these using 4 h doxycycline treatment (Fig. S1), described carried out pulse-chase assays measure kinetics model 1A). BirA fused SNARE Bos1 (BirA-Bos1) because strongly dependent Successful leads glycosylation opsin tag BirA-Bos1 (Cho Shan, 2018), providing quantitative readout efficiency. depletion either did not significantly affect efficiency 1, whereas nearly abolished 1D). exclude possibility defect arose pleiotropic effects tet-YDJ1/tet-SIS1 cells, reduced capacity compromised membrane, tested substrates, Bos1-BirA DHC-αF, whose independent 1A) Bos1-BirA, Bos1-TMD upstream moiety N protein, making co-translational signal particle (SRP) prepro-α replaced aminopeptidase (DAP2) convert SRP-dependent uses (32Ng D.T. Brown J.D. Walter Signal sequences specify route reticulum membrane.J. 134: 269-278Crossref (368) Pulse-chase experiments had only modest E–G) DHC-αF H–J), nonspecific defects insufficient account large deleterious Ydj1/Sis1 BirA-Bos1. It harbors multiple alternative pathways post-translational (33Borgese Coy-Vergara Colombo S.F. Schwappach ways tails: more.Protein 38: 289-305Crossref (39) For example, (SND) mediates internal (34Aviram Ast Costa Arakel E.C. Chuartzman S.G. Jan C.H. Haßdenteufel Dudek Schorr Zimmermann Weissman J.S. SND constitute reticulum.Nature. 540: 134-138Crossref (117) low hydrophobicity GET-independent (35Rao Okreglak Chio Multiple selection filters ensure accurate targeting.Elife. 5e21301Crossref (49) 36Shurtleff Itzhak D.N. Hussmann Schirle Oakdale N.T. Jonikas Weibezahn Popova K.D. Sinitcyn Vembar S.S. Hernandez Cox Burlingame A.L. Brodsky J.L. al.The interacts cotranslationally multipass proteins.Elife. 7e37018Crossref (89) Hsp70/JDP system pathways, BirA-6AG Scs2-GFP S2A). residues Ala/Gly reduce S2B) containing Ydj1/Sis1-dependence cells above. Hsp70-dependence SSA1 (SSA1ssa2Δssa3Δssa4Δ) ssa1ts (ssa1tsssa2Δssa3Δssa4Δ) strains, temperature-sensitive mutant inactivated within 5 min upon shift nonpermissive temperature (37Becker Yan Functional DnaJ-related Ydj1p, vivo.Mol. 4378-4386Crossref (196) While S2C), efficiencies substantially transient inactivation S2, D–G). suggest system, comprised yeast. Together, strong indicate least one them cannot filled form investigated analyses. As soluble autonomously 38Cyr Cooperation specific homologs suppress aggregation.FEBS Lett. 1995; 359: 129-132Crossref (113) 39Lu carboxyl co-chaperone folding.J. 1998; 273: 5970-5978Abstract (151) prevent aggregation. vitro, noncleavable, SUMO (termed Bos1) 35Rao 40Wang Mak Zhuang Denic sorts posttranslational reticulum.Mol. 40: 159-171Abstract (138) Dilution purified, detergent-solubilized aqueous buffer, removes detergent micelles, led monitored turbidity assay 2A, light blue line). Using assay, confirmed effectively prevented (Figs. S3A S1B, red), comparison, ADP-bound efficiently S3, green versus apo-Ssa1 unable blue). form, capture. itself super-stoichiometric concentrations (≥3 μM 1.5 Bos1; Fig. 2A). 0.5 ∼50% 2B, dotted black lines), indicating effective than Bos1. addition substoichiometric amounts enhanced solubility 2, C, cooperates better estimate efficacy Ydj1·Ssa1 capture, sedimentation-based detects (supernatant) insoluble (pellet) fractions D E). absence generated maximum saturating concentrations, >3 saturation 2F, circles). With equ
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ژورنال
عنوان ژورنال: Journal of Biological Chemistry
سال: 2021
ISSN: ['1083-351X', '0021-9258', '1067-8816']
DOI: https://doi.org/10.1016/j.jbc.2021.100546