Polypeptide translocation from the cytosol into the lumen of the endoplasmic reticulum represents the first step in the secretory pathway, and the only event that requires passage of a hydrophilic protein through a lipid bilayer. Recent years have seen a dramatic convergence of observations that suggest a fundamental conservation of the mechanism of protein translocation in prokaryotes and eukaryotes. Work conducted on this grant since the last renewal has led to the discovery of a set of ER membrane proteins and cytosolic hsc7Os that constitute essential elements of the translocation apparatus. One of these molecules, Sec6lp, provides a link between core elements of the E. coli and mammalian polypeptide translocase. Although many of the membrane, cytosolic, and lumenal proteins required for polypeptide import into the ER have now been defined, the exact mechanism of penetration remains obscure. In the previous grant period we developed a method to solubilize yeast membranes and reconstitute translocation in proteoliposomes. One membrane protein complex that contains Sec63p, BiP, and two new Sec proteins was isolated. Additional membrane proteins that are required for translocation are now being isolated with the goal being a fully purified system with which to investigate the nature of the putative channel. Crucial questions of subunit stoichiometry, interactions among the Sec proteins, regulation of channel assembly, and capacity to translocate small molecules will be addressed using Sec proteins isolated from wild type and mutant cells. With a purified set of translocation Sec proteins, we will examine the components that are responsible for signal peptide binding and that are involved in completion of protein import. We have proposed that members of the Sec63p complex interact with the signal peptide portion of presecretory proteins and deliver them to the Sec6lp channel. A subreaction that measures binding of isolated signal peptides to proteoliposomes or soluble membrane proteins is described. A partial reaction that measures BiP-dependent completion of polypeptide transport through the Sec6lp channel will be reproduced in proteoliposomes and in detergent solution. The selective-role of hsc7Os will be examined in the proteoliposome reaction using chimeric molecules made of cytosolic hsc70 and BiP. We hope to define domains of these molecules that confer topological specificity.
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