title: Roles of cytosolic Hsp70 and Hsp40 molecular chaperones in post-translational translocation of presecretory proteins into the endoplasmic reticulum.
abstract: Hsp70 molecular chaperones and their co-chaperones work together in various cellular compartments to guide the folding of proteins and to aid the translocation of proteins across membranes. Hsp70s stimulate protein folding by binding exposed hydrophobic sequences thereby preventing irreversible aggregation. Hsp40s stimulate the ATPase activity of Hsp70s and target unfolded proteins to Hsp70s. Genetic and biochemical evidence supports a role for cytosolic Hsp70s and Hsp40s in the post-translational translocation of precursor proteins into endoplasmic reticulum and mitochondria. To gain mechanistic insight, we measured the effects of Saccharomyces cerevisiae Ssa1p (Hsp70) and Ydj1p (Hsp40) on the translocation of histidine-tagged prepro-alpha-factor (ppalphaF6H) into microsomes. Radiolabeled ppalphaF6H was affinity purified from wheat germ translation reactions (or Escherichia coli) to remove endogenous chaperones. We demonstrated that either Ssa1p or Ydj1p stimulates post-translational translocation by preventing ppalphaF6H aggregation. The binding and/or hydrolysis of ATP by Ssa1p were required to maintain the translocation competence of ppalphaF6H. To clarify the contributions of membrane-bound and cytosolic Ydj1p, we compared the efficiency of chaperone-dependent translocation into wild-type and Ydj1p-deficient microsomes. Neither soluble nor membrane-bound Ydj1p was essential for post-translational protein translocation. The ability of Ssa1p, Ydj1p, or both chaperones to restore the translocation competence of aggregated ppalphaF6H was negligible.
authors.affiliation: Department of Anatomy and Cell Biology, State University of New York Downstate Medical Center, Brooklyn, New York 11203, USA.
authors.forename: Jantra
authors.initials: J
authors.lastname: Ngosuwan
authors.forename: Nancy M
authors.initials: NM
authors.lastname: Wang
authors.forename: Katie L
authors.initials: KL
authors.lastname: Fung
authors.forename: William J
authors.initials: WJ
authors.lastname: Chirico
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000502:physiology*; D006360:Heat-Shock Proteins / Q000502:physiology*; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism; D018832:Molecular Chaperones / Q000502:physiology*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013997:Time Factors; D014158:Transcription, Genetic
chemical_list: D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D029701:Saccharomyces cerevisiae Proteins; C070319:YDJ1 protein, S cerevisiae; D010450:Endopeptidases; D000251:Adenosine Triphosphatases; C095973:SSA1 protein, S cerevisiae; D005033:Ethylmaleimide