The refolding activity of the yeast heat shock proteins Ssa1 and Ssa2 defines their role in protein translocation.
Ssa1/2p, members of one of the yeast cytosolic hsp70 subfamilies, have been implicated in the translocation of secretory proteins into the lumen of the ER. The involvement of these hsp70s in translocation was tested directly by examining the effect of immunodepleting Ssa1/2p from yeast cytosol and subsequently testing the cytosol for its ability to support co- and post-translational translocation of prepro-alpha-factor. Depletion of Ssa1/2p had no effect on the efficiency of translocation in this in vitro assay. The system was used to examine the effect of the absence of Ssa1/2p on two other putative hsp70 functions: cotranslational folding of nascent luciferase and refolding of denatured luciferase. Depletion of Ssa1/2p had no effect on the ability of the yeast lysate to synthesize enzymatically active luciferase, but had a dramatic effect on the ability of the lysate to refold chemically denatured luciferase. These results demonstrate, for the first time, the refolding activity of Ssa1/2p in the context of the yeast cytosol, and define refolding activity as a chaperone function specific to Ssa1/2p, aprt from other cytosolic hsp70s. They also suggest that Ssa1/2p do not play a significant role in chaperoning the folding of nascent polypeptides. The implications of these findings for Ssa1/2p activity on their proposed role in the process of translocation are discussed.
Bush
G L
GL
Department of Biological Chemistry, UCLA School of Medicine 90024-1737, USA.
Meyer
D I
DI
D000251:Adenosine Triphosphatases; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005656:Fungal Proteins / Q000276:immunology / Q000378:metabolism* / Q000502:physiology*; D018840:HSP70 Heat-Shock Proteins / Q000276:immunology / Q000502:physiology*; D008156:Luciferases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D014176:Protein Biosynthesis; D011489:Protein Denaturation; D017510:Protein Folding; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins
D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins; C542288:SSA2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D008156:Luciferases; D000251:Adenosine Triphosphatases; C095973:SSA1 protein, S cerevisiae