Specific molecular chaperone interactions and an ATP-dependent conformational change are required during posttranslational protein translocation intothe yeast ER

The posttranslational translocation of proteins across the endoplasmic reti culum (ER) membrane in yeast requires ATP hydrolysis and the action of hsc7 0s (DnaK homologues) and DnaJ homologues in both the cytosol and ER lumen. Although the cytosolic hsc70 (Ssa1p) and the ER lumenal hsc70 (BiP) are hom ologous, they cannot substitute for one another, possibly because they inte ract with specific DnaJ homologues on each side of the ER membrane. To inve stigate this possibility, we purified Ssa1p, BiP, Ydj1p (a cytosolic DnaJ h omologue), 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-fo ld 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 f rom BiP. To understand further how BiP drives protein translocation, we pur ified four dominant lethal mutants of BiP. We discovered that each mutant i s defective for ATP hydrolysis, fails to undergo an ATP-dependent conformat ional change, and cannot interact with GST-63Jp. Measurements of protein tr anslocation into reconstituted proteoliposomes indicate that the mutants in hibit 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 h sc70 action is dictated by their DnaJ partners.