O-mannosylation protects mutant alpha-factor precursor from endoplasmic reticulum-associated degradation

Secretory proteins that fail to fold in the endoplasmic reticulum (ER) are transported back to the cytosol and degraded by proteasomes. It remains unc lear how the cell distinguishes between folding intermediates and misfolded proteins. We asked whether misfolded secretory proteins are covalently mod ified in the ER before export. We found that a fraction of mutant alpha-fac tor precursor, but not the wild type, was progressively O-mannosylated in m icrosomes and in intact yeast cells by protein O-mannosyl transferase 2 (Pm t2p). O-Mannosylation increased significantly in vitro under ER export cond itions, i.e., in the presence of ATP and cytosol, and this required export- proficient Sec61p in the ER membrane. Deletion of PMT2, however, did not ab rogate mutant alpha-factor precursor degradation but, rather, enhanced its turnover in intact yeast cells. In vitro, O-mannosylated mutant alpha-facto r precursor was stable and protease protected, and a fraction was associate d with Sec61p in the ER lumen. Thus, prolonged ER residence allows modifica tion of exposed O-mannosyl acceptor sites in misfolded proteins, which abro gates misfolded protein export from the ER at a posttargeting stage. We con clude that there is a limited window of time during which misfolded protein s can be removed from the ER before they acquire inappropriate modification s that can interfere with disposal through the Sec61 channel.