Biosynthesis of Lipid-Linked Oligosaccharides in Yeast: the ALG3 Gene Encodes the Dol-P-Man:Man5GlcNAc2-PP-Dol Mannosyltransferase

The formation of Nglycosidic linkages of glycoproteins involves the ordered assembly of the common Glc[3]Man[9]GlcNAc[2] coreoligosaccharide on the lipid carrier dolichyl pyrophosphate. Whereas early mannosylation steps occur on the cytoplasmic side of the endoplasmic reticulum with GDPMan as donor, the final reactions from Man[5]GlcNAc[2]PPDol to Man[9]Glc Nac[2]PPDol on the lumenal side use DolPMan. We have investigated these later stages in vitro using a detergentsolubilized enzyme extract from yeast membranes. Mannosyltransfer from DolPMan to [[3]H]Man[5]GlcNAc[2]PPDol with formation of all intermediates up to Man[9]GlcNAc[2]PPDol occured in a rapid, time and proteindependent fashion. We find that the initial reaction from Man[5]GlcNAc[2]PPDol to Man[6]GlcNAc[2]PPDol is independent of metal ions, but further elongations need Mn[2+] that can be partly replaced by Mg[2+] or Ca[2+]. Zn[2+] or Cd[2+] ions were found to inhibit formation of Man[7-9]GlcNAc[2]PPDol, but do not affect synthesis of Man[6]GlcNAc[2]PPDol. Extension did not occur when the acceptor was added as a free Man[5]GlcNAc[2] oligosaccharide or when GDPMan was used as mannosyldonor. The alg3 mutant was described to accumulate Man[5]GlcNAc[2]PPDol. We expressed a functional active HAepitope tagged ALG3 fusion and succeeded to selectively immunoprecipitate the DolPMan:Man[5]GlcNAc[2]PPDol mannosyltransferase activity from the other enzymes of the detergent extract involved in the subsequent mannosylation reactions. This demonstrates that Alg3p represents the mannosyltransferase itself and not an accessory protein involved in the reaction.