Assembly of ER-associated protein degradation in vitro: dependence on cytosol, calnexin, and ATP

• Published in: The Journal of cell biology 1996-02, Vol.132 (3), p.291-298
• Main Authors: McCracken, Ardythe A., Brodsky, Jeffrey L.
• Format: Article
• Language: English
• Subjects: adenosine triphosphate; Adenosine Triphosphate - metabolism; adenosintrifosfato; binding proteins; Biological Transport; Calcium-Binding Proteins - metabolism; Calnexin; Cell biology; Cell free system; Cellular biology; citoplasma; cytoplasm; cytoplasme; cytoplasmic organelles; Cytosol; Cytosol - metabolism; degradacion; degradation; Endoplasmic reticulum; Endoplasmic Reticulum - metabolism; feromonas; Fungal Proteins - metabolism; Glycoproteins - metabolism; Material degradation; Mating Factor; Microsomes; Microsomes - metabolism; Molecular chaperones; organite cellulaire; organulos citoplasmicos; Peptides - metabolism; pheromone; pheromones; Protein Precursors - metabolism; Protein Processing, Post-Translational; Protein transport; proteinas; proteinas aglutinantes; proteine; proteine de liaison; Proteins; reticulo endoplasmatico; reticulum endoplasmique; saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; T lymphocytes; Yeast; Yeasts
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.132.3.291

To investigate the mechanisms of ER-associated protein degradation (ERAD), this process was reconstituted in vitro. Established procedures for posttranslational translocation of radiolabeled prepro-alpha factor into isolated yeast microsomes were modified to inhibit glycosylation and to include a posttranslocation "chase" incubation period to monitor degradation. Glycosylation was inhibited with a glyco-acceptor peptide to compete for core carbohydrates, or by using a radiolabeled alpha factor precursor that had been genetically engineered to eliminate all three glycosylation sites. Inhibition of glycosylation led to the production of unglycosylated pro-alpha factor (palphaF), a processed form of the alpha factor precursor shown to be a substrate of ERAD in vivo. With this system, both glycosylated and unglycosylated forms of pro-alpha factor were stable throughout a 90-min chase incubation. However, the addition of cytosol to the chase incubation reaction induced a selective and rapid degradation of palphaF. These results directly reflect the behavior of alpha factor precursor in vivo; i.e., palphaF is a substrate for ERAD, while glycosylated pro-alpha factor is not. Heat inactivation and trypsin treatment of cytosol, as well as addition of ATPgammaS to the chase incubations, led to a stabilization of palphaF. ERAD was observed in sec12 microsomes, indicating that export of palphaF via transport vesicles was not required. Furthermore, palphaF but not glycosylated pro-alpha factor was found in the supernatant of the chase incubation reactions, suggesting a specific transport system for this ERAD substrate. Finally, the degradation of palphaF was inhibited when microsomes from a yeast strain containing a disrupted calnexin gene were examined. Together, these results indicate that cytosolic protein factor(s), ATP hydrolysis, and calnexin are required for ER-associated protein degradation in yeast, and suggest the cytosol as the site for degradation.