Functional Interaction of Cytosolic hsp70 and a DnaJ-Related Protein, Ydj1p, in Protein Translocation In Vivo

Abstract

In order to analyze the in vivo role of the SSA class of cytosolic 70-kDa heat shock proteins (hsps) of Saccharomyces cerevisiae, we isolated a temperature-sensitive mutant of SSA1. The effect of a shift of mutant cells (ssa1 ts ssa2 ssa3 ssa4) from the permissive temperature of 23؇C to the nonpermissive temperature of 37؇C on the processing of several precursor proteins translocated into the endoplasmic reticulum or mitochondria was assessed. Of three mitochondrial proteins tested, the processing of only one, the ␤ subunit of the F 1 F 0 ATPase, was dramatically affected. Of six proteins destined for the endoplasmic reticulum, the translocation of only prepro-␣-factor and proteinase A was inhibited. The processing of prepro-␣-factor was inhibited within 2 min of the shift to 37؇C, suggesting a direct effect of the hsp70 defect on translocation. More than 50% of radiolabeled ␣-factor accumulated in the precursor form, with the remainder rapidly reaching the mature form. However, the translocation block was complete, as the precursor form could not be chased through the translocation pathway. Since DnaJ-related proteins are known to interact with hsp70s and strains containing conditional mutations in a dnaJ-related gene, YDJ1, are defective in translocation of prepro-␣-factor, we looked for a genetic interaction between SSA genes and YDJ1 in vivo. We found that a deletion mutation of YDJ1 was synthetically lethal in a ssa1 ts ssa2 ssa3 ssa4 background. In addition, a strain containing a single functional SSA gene, SSA1, and a deletion of YDJ1 accumulated the precursor form of ␣-factor. However, no genetic interaction was observed between a YDJ1 mutation and mutations in the SSB genes, which encode a second class of cytosolic hsp70 chaperones. These results are consistent with SSA proteins and Ydj1p acting together in the translocation process.

Chaperone action in the posttranslational topological reorientation of the hepatitis B virus large envelope protein: Implications for translocational regulation

“…The lack of chaperone binding of M and S coincides with their classical cotranslational folding pathway and hence provides evidence for functional roles of Hsc70 and BiP in controlling the nonclassical translocation of L. In support, we observed that artificial enforcement of cotranslational preS translocation of L blocked its association with Hsc70, implicating this chaperone to be responsible for the suppression of the cotranslational preS import into the ER. Our finding that Hsp40 participates in Hsc70-L complex formation is further suggestive of a productive interaction between L and Hsc70, because members of the Hsp40 family are required for Hsc70 function (20,30). In this respect, L shares features with the cystic fibrosis transmembrane conductance regulator, a polytopic membrane protein that also exposes large subdomains to the cytosolic side of the ER membrane where those are held and folded by the Hsp40͞Hsc70 chaperone pair (31).…”

“…Binding to L. To productively guide translocating proteins across membranes, Hsc70 must interact with co-chaperones, such as with the human DnaJ family protein Hsp40, that regulate its ability to bind and release substrate polypeptides (20,27). Therefore, we inspected the L-Hsc70 complex for the presence of Hsp40 (Hdj-1).…”

“…Posttranslational protein translocation into the ER is found in all eukaryotic cells (15, 16), but so far this pathway has been best studied in yeast where it involves the heptameric Sec complex plus cytosolic and luminal chaperones of the heat shock protein (Hsp) Hsp70 ATPase family (16)(17)(18)(19). Cytosolic Hsp70s, such as the cognate member Hsc70, in cooperation with Hsp40 class molecules, stimulate the import of proteins into the ER (17,18,20) whereas the luminal Hsp70, the BiP protein (in yeast: Kar2p) is required to provide the driving force for the vectorial movement of polypeptides into the ER lumen (16,19,21). During our search for cellular factors potentially involved in regulating L topogenesis, we previously identified Hsc70 as a specific binding partner of the preS1 domain in vitro (22).…”

The Hsp110 protein chaperone Sse1 is required for yeast cell wall integrity and morphogenesis

“…The ssa1-45 allele of SSA1 is inactivated at high temperature, and in the absence of the partially redundant SSA2, SSA3 and SSA4 genes results in inviability at 37°C (Becker et al 1996). Growth of this mutant at the restrictive temperature, or a strain lacking both SSE1 and SSE2, was not restored in the presence of salt or sorbitol, demonstrating that osmotic stabilization does not indiscriminately rescue loss of global chaperone function, but rather compensates for a specific defect in cell integrity signaling (Fig.…”

“…The SLT2-13xmyc strain (BY2207) and isogenic wild type were generous gifts of B. Andrews (University of Toronto). The ssa1-45 allele and the isogenic wild type strain JN516 were kind gifts of E. Craig (University of Wisconsin) (Becker et al 1996). The FES1 ORF was subcloned via SpeI and XhoI sites from p413GPDFES1 into plasmid p423GPD to create p423GPDFES1 (Shaner et al 2006).…”