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Vol. 9, Issue 12, 3533-3545, December 1998
and
*Department of Biological Sciences, University of Pittsburgh,
Pittsburgh, Pennsylvania 15260; and
The posttranslational translocation of proteins across the
endoplasmic reticulum (ER) membrane in yeast requires ATP
hydrolysis and the action of hsc70s (DnaK homologues) and DnaJ
homologues in both the cytosol and ER lumen. Although the cytosolic
hsc70 (Ssa1p) and the ER lumenal hsc70 (BiP) are homologous, they
cannot substitute for one another, possibly because they interact with specific DnaJ homologues on each side of the ER membrane. To
investigate this possibility, we purified Ssa1p, BiP, Ydj1p (a
cytosolic DnaJ homologue), and a GST-63Jp fusion protein containing
the lumenal DnaJ region of Sec63p. We observed that BiP, but not
Ssa1p, is able to associate with GST-63Jp and that Ydj1p stimulates
the ATPase activity of Ssa1p up to 10-fold but increases the ATPase activity of BiP by <2-fold. In addition, Ydj1p and ATP trigger the
release of an unfolded polypeptide from Ssa1p but not from BiP.
To understand further how BiP drives protein translocation, we
purified four dominant lethal mutants of BiP. We discovered that each
mutant is defective for ATP hydrolysis, fails to undergo an
ATP-dependent conformational change, and cannot interact with GST-63Jp. Measurements of protein translocation into reconstituted proteoliposomes indicate that the mutants inhibit translocation even in
the presence of wild-type BiP. We conclude that a conformation- and
ATP-dependent interaction of BiP with the J domain of Sec63p is
essential for protein translocation and that the specificity of hsc70
action is dictated by their DnaJ partners.
Department of
Molecular Biology, Princeton University, Princeton, New Jersey 08544
Corresponding author: Department of
Biological Sciences, University of Pittsburgh, 267 Crawford Hall,
Pittsburgh, PA 15260. E-mail address: jbrodsky+{at}pitt.edu.
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