The accumulation of Man6GlcNAc2-PP-dolichol in the Saccharomyces cerevisiae Δalg9 mutant reveals a regulatory role for the Alg3p α1,3-man middle-arm addition in downstream oligosaccharide-lipid and glycoprotein glycan processing

N-Glycans in nearly all eukaryotes are derived by transfer of a precursor Glc(3)Man(9)GlcNAc(2) from dolichol (Dol) to consensus Asn residues in nascent proteins in the endoplasmic reticulum, The Saccharomyces cerevisiae alg (asparagine-linked glycosylation) mutants fail tb synthesize oligosaccharide-lipid properly, and the alg9 mutant, accumulates Man(6)GlcNAc(2)-PP-Dol. Highfield H-1 NMR and methylation analyses of Man(6)GlcNAc(2) released with peptide-N-glycosidase F from invertase secreted by Delta alg9 yeast showed its structure to be Man alpha 1,2Man alpha 1,2Man alpha 1,3(Man alpha 1,3Man alpha 1,6)-Man beta 1, 4GlcNAc beta 1,4GlcNAc alpha/beta, confirming the addition of the alpha 1,3-linked Man to Man(5)GlcNAc(2)-PP-Dol prior to the addition of the final upper-arm al,g-linked Man. This Man(6)GlcNAc(2), is the endoglycosidase H-sensitive product of the Alg3p step. The Delta alg9 Hex7-10GlcNAc(2) elongation intermediates were released from invertase and similarly analyzed. When compared with alg3 sec18 and wild-type core mannans, Delta alg9 N-glycans reveal a regulatory role for the Alg3p-dependent alpha 1,3-linked Man in subsequent oligosaccharide-lipid and glycoprotein glycan maturation. The presence of this Man appears to provide structural information potentiating the downstream action of the endoplasmic reticulum glucosyltransferases Alg6p, Alg8p and Alg10p, glucosidases Gls1p and Gls2p, and the Golgi Och1p outerchain alpha 1,6-Man branch-initiating mannosyltransferase.