UBIQUITINATION IS REQUIRED FOR THE RETRO-TRANSLOCATION OF A SHORT-LIVED LUMINAL ENDOPLASMIC-RETICULUM GLYCOPROTEIN TO THE CYTOSOL FOR DEGRADATION BY THE PROTEASOME

In the endoplasmic reticulum (ER), an efficient ''quality control syst em'' operates to ensure that mutated and incorrectly folded proteins a re selectively degraded. We are studying ER-associated degradation usi ng a truncated variant of the rough ER-specific type I transmembrane g lycoprotein, ribophorin I. The truncated polypeptide (RI332) consists of only the 332 amino-terminal amino acids of the protein correspondin g to most of its luminal domain and, in contrast to the long-lived end ogenous ribophorin I, is rapidly degraded. Here we show that the ubiqu itin-proteasome pathway is involved in the destruction of the truncate d ribophorin I. Thus, when RI332 that itself appears to be a substrate for ubiquitination was expressed in a mutant hamster cell line harbor ing a temperature-sensitive mutation in the ubiquitin-activating enzym e El affecting ubiquitin dependent proteolysis, the protein is dramati cally stabilized at the restrictive temperature. Moreover, inhibitors of proteasome function effectively block the degradation of RI332. Cel l fractionation experiments indicate that RI332 accumulates in the cyt osol when degradation is prevented by proteasome inhibitors but remain s associated with the lumen of the ER under ubiquitination deficient c onditions, suggesting that the release of the protein into the cytosol is ubiquitination-dependent. Accordingly, when ubiquitination is impa ired, a considerable amount of RI332, binds to the ER chaperone calnex in and to the Sec61 complex that could effect retro-translocation of t he polypeptide to the cytosol, Before proteolysis of RI332, its N-link ed oligosaccharide is cleaved in two distinct steps, the first of whic h might occur when the protein is still associated with the ER, as the trimmed glycoprotein intermediate efficiently interacts with calnexin and Sec61, From our data we conclude that the steps that lead a newly synthesized luminal ER glycoprotein to degradation by the proteasome are tightly coupled and that especially ubiquitination plays a crucial role in the retro-translocation of the substrate protein for proteoly sis to the cytosol.