Reconstitution of protein translocation from solubilized yeast membranes reveals topologically distinct roles for BiP and cytosolic Hsc70.
We reconstituted prepro-alpha-factor translocation 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 observed with vesicles reconstituted from a mixture of pure wild-type yeast BiP and a soluble fraction of kar2 mutant membranes. Two other heat-shock cognate (hsc) 70 homologs, yeast cytosolic hsc70 (Ssalp) and E. coli dnaK protein did not replace BiP. Conversely, BiP was not active under conditions where translocation into native ER vesicles required cytosolic hsc70. We conclude that cytosolic hsc70 and BiP serve noninterchangeable roles in polypeptide translocation, possibly because distinct, asymmetrically oriented membrane proteins are required to recruit each protein to opposing surfaces of the ER membrane.
Brodsky
J L
JL
Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley 94720.
Hamamoto
S
S
Feldheim
D
D
Schekman
R
R
D001692:Biological Transport; D002474:Cell-Free System; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D029968:Escherichia coli Proteins; D005656:Fungal Proteins / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism; D000072235:Mating Factor; D008854:Microscopy, Electron; D010455:Peptides / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism; D012441:Saccharomyces cerevisiae
D029968:Escherichia coli Proteins; D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C060153:KAR2 protein, yeast; D010455:Peptides; D021382:Protein Sorting Signals; D000072235:Mating Factor; C063842:dnaK protein, E coli