Slslp stimulates Sec63p-mediated activation of Kar2p in a conformation-dependent manner in the yeast endoplasmic reticulum

We previously characterized the SLS1 gene in the yeast Yarrowia lipolytica and showed that it interacts physically with YlKar2p to promote translocati on across the endoplasmic reticulum membrane (A. Boisrame, M. Kahani, J. M. Beckerich, E. Hartmann, and C. Gaillardin, J. Biol. Chem, 273:30903-30908, 1998). A Y. lipolytica Kar2p mutant was isolated that restored interaction with an Sls1p mutant, suggesting that the interaction with Sls1p could be nucleotide and/or conformation dependent. This result was used as a working hypothesis for more accurate investigations in Saccharomyces cerevisiae. W e show by two-hybrid an in vitro assays that the S. cerevisiae homologue of Sls1p interacts with ScKar2p. Using dominant lethal mutants of ScKar2p, we were able to show that ScSls1p preferentially interacts with the ADP-bound conformation of the molecular chaperone. Synthetic lethality was observed between Delta Scsls1 and translocation-deficient kar2 or sec63-1 mutants, p roviding in vivo evidence for a role of ScSls1p in protein translocation. S ynthetic lethality was also observed with ER-associated degradation and fol ding-deficient kar2 mutants, strongly suggesting that Sls1p functions are n ot restricted to the translocation process. We show that Sls1p stimulates i n a dose-dependent manner the binding of ScKar2p on the lumenal J domain of Sec63p fused to glutathione S-transferase. Moreover, Sls1p is shown to pro mote the Sec63p-mediated activation of Kar2p's ATPase activity. Our data st rongly suggest that Sls1p could be the first GrpE-like protein described in the endoplasmic reticulum.