The Genetic Dissection of Protein Translocation Across the Yeast ER Membrane

Abstract

The first stage in the eukaryotic secretory pathway is the translocation of polypeptides across the endoplasmic reticulum (ER) membrane. This process has been extensively studied in mammalian systems using an in vitro assay which faithfully reproduces the cotranslational translocation of specific precursor proteins into the ER lumen (Blobel and Dobberstein, 1975). Biochemical analysis of the components required to support translocation, has revealed requirements for both cytosolic, and membrane associated factors. Of these, the best characterised to date is signal recognition particle (SRP), a cytosolic ribonucleoprotein complex comprising six polypeptide sub-units (9, 14, 19, 54, 68, 72kDa), plus one molecule of 7SL RNA (Walter & Blobel, 1980; Walter & Blobel, 1982). Evidence suggests that SRP binds to the signal sequence of a nascent secretory protein as it emerges from the ribosome. The binding of SRP to the nascent chain/ribosome complex significantly reduces the rate of polypeptide chain elongation (elongation arrest, Walter and Blobel, 1981). The arrested complex is then targeted to the ER membrane via the interaction of SRP with an integral membrane G-protein known as SRP receptor, or “docking protein”, (Gilmore et al., 1982; Meyer et al., 1982). The SRP-receptor mediates the GTP-dependent displacement of SRP from the signal sequence/ribosome complex (Connolly & Gilmore, 1989). The displacement of SRP relieves elongation arrest, thus permitting the targeted precursor to be co-translationally translocated across the ER membrane.