Zusammenfassung
The formation of N-glycosidic linkages of glycoproteins involves the ordered assembly of the common Glc(3)Man(9)GlcNAc(2) core-oligosaccharide on the lipid carrier dolichyl pyrophosphate. Whereas early mannosylation steps occur on the cytoplasmic side of the endoplasmic reticulum with GDP-Man as donor, the final reactions from Man(5)GlcNAc(2)-PP-Dol to Man(6)GlcNAc(2)-PP-Dol on the lumenal side ...
Zusammenfassung
The formation of N-glycosidic linkages of glycoproteins involves the ordered assembly of the common Glc(3)Man(9)GlcNAc(2) core-oligosaccharide on the lipid carrier dolichyl pyrophosphate. Whereas early mannosylation steps occur on the cytoplasmic side of the endoplasmic reticulum with GDP-Man as donor, the final reactions from Man(5)GlcNAc(2)-PP-Dol to Man(6)GlcNAc(2)-PP-Dol on the lumenal side use Dol-P-Man. We have investigated these later stages in vitro using a detergent-solubilized enzyme extract from yeast membranes. Mannosyltransfer from Dol-P-Man to [H-3]Man(5)GlcNAc(2)-PP-Dol with formation 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 from 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)GlcNAc(2)-PP-Dol, but do not affect synthesis of Man(6)GlcNAc(2)-PP-Dol. Extension did not occur when the acceptor was added as a free Man(5)GlcNAc(2) oligosaccharide or when GDP-Man was used as mannosyldonor. The alg3 mutant was described to accumulate Man(5)GlcNAc(2)-PP-Dol. We expressed a functional active HA-epitope tagged ALG3 fusion and succeeded to selectively immunoprecipitate 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 mannosyltransferase itself and not an accessory protein involved in the reaction.
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