FOLDING, UNFOLDING, AND REFOLDING OF THE VESICULAR STOMATITIS-VIRUS GLYCOPROTEIN

Folding and refolding of the vesicular stomatitis virus (VSV) glycopro tein (G protein), New Jersey serotype, were studied both in infected c ells and after urea denaturation and reduction of isolated protein in vitro. To assess the contribution of disulfide bonds to the conformati on of this type I membrane glycoprotein, reduced and alkylated forms w ere compared with unreduced G proteins by their mobility on SDS-polyac rylamide gels and by their reactivity with conformation-dependent mono clonal antibodies (MAbs). Pulse-chase experiments showed that G protei n folding in the endoplasmic reticulum (ER) of infected cells occurred rapidly (estimated half-time of 1-2 min) and involved transient assoc iation with the ER chaperone calnexin, Inhibition of glycosylation by tunicamycin slowed the folding process and emergence from the ER but d id not prevent the appearance of a conformationally mature transport-c ompetent G protein, For in vitro refolding studies, native G protein i solated from virus particles was denatured and reduced with urea and b eta-mercaptoethanol. When rapidly diluted into a denaturant-free buffe r containing oxidized glutathione and the nonionic detergent octyl glu coside. the Cr protein regained considerable native structure, as dete rmined by reactivity with five monoclonal antibodies: specific for dif ferent conformation-dependent epitopes. Whereas the refolding process was slow and inefficient in vitro relative to folding in the cell, thi s observation nonetheless demonstrated that an integral fully glycosyl ated membrane protein can be refolded to form a structure similar to t hat of the original protein processed during in vitro synthesis, If, h owever, unfolded nonglycosylated G protein was the starting material, refolding in vitro failed. In summary, we have shown that VSV G protei n folding can be analyzed both in vitro and in vitro and that folding in the cell involves at least one chaperone and can occur in vitro eve n if not glycosylated.