Folding, Unfolding, and Refolding of the Vesicular Stomatitis Virus Glycoprotein
Folding and refolding of the vesicular stomatitis virus (VSV) glycoprotein (G protein), New Jersey serotype, were studied both in infected cells and after urea denaturation and reduction of isolated protein in vitro. To assess the contribution of disulfide bonds to the conformation of this type I me...
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Published in | Biochemistry (Easton) Vol. 35; no. 13; pp. 4084 - 4093 |
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Main Authors | , , , |
Format | Journal Article |
Language | English |
Published |
United States
American Chemical Society
02.04.1996
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Subjects | |
Online Access | Get full text |
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Summary: | Folding and refolding of the vesicular stomatitis virus (VSV) glycoprotein (G protein), New Jersey serotype, were studied both in infected cells and after urea denaturation and reduction of isolated protein in vitro. To assess the contribution of disulfide bonds to the conformation of this type I membrane glycoprotein, reduced and alkylated forms were compared with unreduced G proteins by their mobility on SDS−polyacrylamide gels and by their reactivity with conformation-dependent monoclonal antibodies (MAbs). Pulse−chase experiments showed that G protein folding in the endoplasmic reticulum (ER) of infected cells occurred rapidly (estimated half-time of 1−2 min) and involved transient association with the ER chaperone calnexin. Inhibition of glycosylation by tunicamycin slowed the folding process and emergence from the ER but did not prevent the appearance of a conformationally mature transport-competent G protein. For in vitro refolding studies, native G protein isolated from virus particles was denatured and reduced with urea and β-mercaptoethanol. When rapidly diluted into a denaturant-free buffer containing oxidized glutathione and the nonionic detergent octyl glucoside, the G protein regained considerable native structure, as determined by reactivity with five monoclonal antibodies specific for different conformation-dependent epitopes. Whereas the refolding process was slow and inefficient in vitro relative to folding in the cell, this observation nonetheless demonstrated that an integral fully glycosylated membrane protein can be refolded to form a structure similar to that of the original protein processed during in vivo synthesis. If, however, unfolded nonglycosylated G protein was the starting material, refolding in vitro failed. In summary, we have shown that VSV G protein folding can be analyzed both in vivo and in vitro and that folding in the cell involves at least one chaperone and can occur in vivo even if not glycosylated. |
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Bibliography: | istex:33E4E0A2AE739D8BA7166B9096B812497A2539C5 ark:/67375/TPS-073TKHZF-8 This research was supported by Grants R37 AI-11112, GM38346, and CA46128 from the National Institutes of Health and by Grant 3158 from the Council for Tobacco Research. M.E.M. was supported partly by a grant from the Robert Wood Johnson Foundation under the Minority Medical Faculty Development Program. Abstract published in Advance ACS Abstracts, March 15, 1996. ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2 |
ISSN: | 0006-2960 1520-4995 |
DOI: | 10.1021/bi952924i |