Roles of cytosolic Hsp70 and Hsp40 molecular chaperones in post-translational translocation of presecretory proteins into the endoplasmic reticulum

Jantra Ngosuwan, Nancy M. Wang, Katie L. Fung, William J. Chirico

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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 preproα-factor (ppαF6H) into microsomes. Radiolabeled ppαF6H 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 ppaF6H aggregation. The binding and/or hydrolysis of ATP by Ssa1p were required to maintain the translocation competence of ppαF6H. 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 ppαF6H was negligible.

Original languageEnglish
Pages (from-to)7034-7042
Number of pages9
JournalJournal of Biological Chemistry
Volume278
Issue number9
DOIs
Publication statusPublished - 2003 Feb 28

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All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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