J-domain proteins promote client relay from Hsp70 during tail-anchored membrane protein targeting

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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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. 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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