Reconstimtion of Protein Translocation from Solubilized Yeast Membranes Reveals Topologically Distinct Roles

Abstract. We reconstituted prepro-ot-factor transloca-tion and signal peptide processing using a yeast microsomal detergent soluble fraction formed into vesicles with soybean phospholipids. Reconstituted translocation required ATP, and was deficient when sec63 and kar2 (BiP) mutant cells were used as a source of membranes. Normal translocation was ob-served with vesicles reconstituted from a mixture of pure wild-type yeast BiP and a soluble fraction of kar2 mutant membranes. Two other heat-shock cog-nate (hsc)70 homologs, yeast eytosolic hsc70 (Ssalp) and E. coli dnaK protein did not replace BiP. Con-versely, BiP was not active under conditions where translocation i to native ER vesicles required eytosolic hse70. We conclude that cytosolic hse'/0 and BiP serv