The yeast SEC20 gene is required for N- and O-glycosylation in the Golgi -Evidence that impaired glycosylation does not correlate with the secretorydefect

The Golgi plays a fundamental role in posttranslational glycosylation, tran sport, and sorting of proteins. The mechanism of protein transport through the Golgi has been seen as controversial in recent years. During the charac terization of N-glycosylation-defective mutants (ngd) previously isolated b y this laboratory, it was found that ngd20 is allelic to sec20. SEC20 was r eported to be required for transport from endoplasmic reticulum to Golgi, b ut its precise function remains to be determined. We show now that SEC20 is also required for N- and O-glycosylation in the Golgi but not in the Ell, in a cargo-specific manner, and that the glycosylation defect does not corr elate with the secretory defect. By pulse-chase labeling experiments in com bination with mannose linkage-specific antibodies, invertase and carboxypep tidase were found to be efficiently secreted to their final compartment, ev en upon shift to the nonpermissive temperature, while glycosylation in the Golgi was severely impaired. Using microsomal membranes isolated from ngd20 , we found that mannosyl transfer from GDP-Man to various mannose-oligosacc harides, indicative for Golgi mannosylation, was strongly diminished. Analy sis of the carbohydrate component of chitinase, an exclusively O-mannosylat ed protein, or of the bulk mannoprotein indicates that O-mannosylation is a lso reduced. The results demonstrate that in addition to secretion SEC20 al so affects glycosylation in the Golgi, presumably because it exerts a more general role in maintenance and function of the Golgi compartments.