Biosynthesis of lipid-linked oligosaccharides in yeast: the ALG3 gene encodes the DoI-P-Man : Man(5)GlcNAc(2)-PP-DoI mannosyltransferase

The formation of N-glycosidic linkages of glycoproteins involves the ordere d assembly of the common Glc(3)Man(9)GlcNAc(2) core-oligosaccharide on the lipid carrier dolichyl pyrophosphate. Whereas early mannosylation steps occ ur on the cytoplasmic side of the endoplasmic reticulum with GDP-Man as don or, the final reactions from Man(5)GlcNAc(2)-PP-Dol to Man(6)GlcNAc(2)-PP-D ol on the lumenal side use Dol-P-Man. We have investigated these later stag es in vitro using a detergent-solubilized enzyme extract from yeast membran es. Mannosyltransfer from Dol-P-Man to [H-3]Man(5)GlcNAc(2)-PP-Dol with for mation of all intermediates up to Man(9)GlcNAc(2)-PP-Dol occured in a rapid , time- and protein-dependent fashion. We find that the initial reaction fr om Man(5)GlcNAc(2)-PP-Dol to Man(6)GlcNAc(2)-PP-Dol is independent of metal ions, but further elongations need Mn2+ that can be partly replaced by Mg2 + or Ca2+. Zn2+ or Cd2+ ions were found to inhibit formation of Man(7-9)Glc NAc(2)-PP-Dol, but do not affect synthesis of Man(6)GlcNAc(2)-PP-Dol. Exten sion did not occur when the acceptor was added as a free Man(5)GlcNAc(2) ol igosaccharide or when GDP-Man was used as mannosyldonor. The alg3 mutant wa s described to accumulate Man(5)GlcNAc(2)-PP-Dol. We expressed a functional active HA-epitope tagged ALG3 fusion and succeeded to selectively immunopr ecipitate the Dol-P-Man:Man(5)GlcNAc(2)-PP-Dol mannosyltransferase activity from the other enzymes of the detergent extract involved in the subsequent mannosylation reactions. This demonstrates that Alg3p represents the manno syltransferase itself and not an accessory protein involved in the reaction .