| Title: | "Assembly of ER-associated protein degradation in vitro: dependence on cytosol, calnexin, and ATP" |
| Author(s): | McCracken AA; Brodsky JL; |
| Address: | "University of Nevada, Biology Department, Reno 89557, USA. Ardythe_McCracken.BioChem@muwaye.unimelb.edu.au" |
| ISSN/ISBN: | 0021-9525 (Print) 1540-8140 (Electronic) 0021-9525 (Linking) |
| Abstract: | "To investigate the mechanisms of ER-associated protein degradation (ERAD), this process was reconstituted in vitro. Established procedures for post-translational 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 radio-labeled 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 (p alpha F), 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 p alpha F. These results directly reflect the behavior of alpha factor precursor in vivo; i.e., p alpha F is a substrate for ERAD, while glycosylated pro-alpha factor is not. Heat inactivation and trypsin treatment of cytosol, as well as addition of ATP gamma S to the chase incubations, led to a stabilization of p alpha F. ERAD was observed in sec12 microsomes, indicating that export of p alpha F via transport vesicles was not required. Furthermore, p alpha F 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 p alpha F 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" |
| Keywords: | Adenosine Triphosphate/*metabolism Biological Transport Calcium-Binding Proteins/*metabolism Calnexin Cytosol/metabolism Endoplasmic Reticulum/*metabolism Fungal Proteins/*metabolism Glycoproteins/metabolism Mating Factor Microsomes/metabolism Peptides/me; |
| Notes: | "MedlineMcCracken, A A Brodsky, J L eng Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. 1996/02/01 J Cell Biol. 1996 Feb; 132(3):291-8. doi: 10.1083/jcb.132.3.291" |
