Aberrant polypeptides in the endoplasmic reticulum (ER) are retro-translocated to the cytoplasm and degraded by the 26S proteasome via ER-associated degradation (ERAD). To begin to resolve the requirements for the retro-translocation and degradation steps during ERAD, a cell-free assay was used to investigate the contributions of specific factors in the yeast cytosol and in ER-derived microsomes during the ERAD of a model, soluble polypeptide. As ERAD was unaffected when cytoplasmic chaperone activity was compromised, we asked whether proteasomes on their own supported both export and degradation in this system. Proficient ERAD was observed if wild-type cytosol was substituted with either purified yeast or mammalian proteasomes. Moreover, addition of only the 19S cap of the proteasome catalyzed ATPdependent export of the polypeptide substrate, which was degraded upon subsequent addition of the 20S particle.
Secretory proteins that fail to fold in the endoplasmic reticulum (ER) are transported back to the cytosol and degraded by proteasomes. It remains unclear how the cell distinguishes between folding intermediates and misfolded proteins. We asked whether misfolded secretory proteins are covalently modified in the ER before export. We found that a fraction of mutant alpha-factor precursor, but not the wild type, was progressively O-mannosylated in microsomes and in intact yeast cells by protein O-mannosyl transferase 2 (Pmt2p). O-Mannosylation increased significantly in vitro under ER export conditions, 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 abrogate mutant alpha-factor precursor degradation but, rather, enhanced its turnover in intact yeast cells. In vitro, O-mannosylated mutant alpha-factor precursor was stable and protease protected, and a fraction was associated with Sec61p in the ER lumen. Thus, prolonged ER residence allows modification of exposed O-mannosyl acceptor sites in misfolded proteins, which abrogates misfolded protein export from the ER at a posttargeting stage. We conclude that there is a limited window of time during which misfolded proteins can be removed from the ER before they acquire inappropriate modifications that can interfere with disposal through the Sec61 channel.
BackgroundThe split-ubiquitin system monitors interactions of transmembrane proteins in yeast. It is based on the formation of a quasi-native ubiquitin structure upon interaction of two proteins to which the N- and C-terminal halves of ubiquitin have been fused. In the system we use here ubiquitin formation leads to proteolytic cleavage liberating a transcription factor (PLV) from the C-ubiquitin (C) fusion protein which can then activate reporter genes. Generation of fusion proteins is, however, rife with problems, and particularly in transmembrane proteins often disturbs topology, structure and function.ResultsWe show that both the Sec61 protein which forms the principal protein translocation channel in the endoplasmic reticulum (ER) membrane, and its non-essential homologue, Ssh1p, when fused C-terminally to CPLV are inactive. In a heterozygous diploid Sec61-CPLV is present in protein translocation channels in the ER membrane without disturbing their function and displays a limited set of protein-protein interactions similar to those found for the wildtype protein using biochemical methods. Although its expression level is similar, Ssh1-CPLV interactions are less strong, and, in contrast to Sec61p, Ssh1p does not distinguish between Sbh1p and Sbh2p. We show that interactions can be monitored by reporter gene activity or directly by PLV cleavage, which is more sensitive, but leads to quantitatively different results.ConclusionsWe conclude that the split-ubiquitin system we used here has high fidelity, but low sensitivity and is of limited use for detection of new, transient interactions with protein translocation channels in the ER membrane.
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