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PubMed:8608269 / 1247-1258 JSONTXT

Mannosyltransferase activities in membranes from various yeast strains. In the yeast Golgi compartments, at least five, and potentially several additional mannosyltransferases are involved in elongating to 'mannan' the core Man8GlcNAc2 oligosaccharide trimmed from Glc3Man9GlcNAc2 in the endoplasmic reticulum. Structural studies on oligosaccharides from alg3 mutant yeast, which lack the four upper arm mannoses donated by Man-P-Dol (where Dol is dolichol), verified that the new alpha 1,6-branch in endo H-resistant mannan in this strain is efficiently initiated in vivo on the alpha 1,3-linked core residue of the lipid-oligosaccharide form of Man5GlcNAc2 (Verostek et al., J. Biol. Chem., 266, 5547-5551, 1991). This Man5GlcNAcGlcNAc[3H]ol isomer (where GlcNAc[3H]ol is N-acetylglucosamin [1-3H] itol) was found to be an excellent acceptor for a number of GDP-Man-dependent Golgi mannosyltransferases in detergent-solubilized yeast membrane preparations: an alpha 1,3-mannosyltransferase (Mnn1p), an alpha 1,6-mannosyltransferase (Och1p) and two alpha 1,2-mannosyltransferases (Mnt1p/Kre2p,?) whose products were readily identified by 1H NMR spectroscopy. The Man6GlcNAcGlcNAc[3H]ol isomers formed were easily defined by alpha 1,2-mannosidase sensitivity and either Bio-Gel P-4 gel filtration or AX-5 high-performance liquid chromatography. In general, mannosyltransferases present in detergent-solubilized microsomes from most yeast strains mimicked the array of sugar linkages observed on their respective glycoproteins. However, in the case of the Saccharomyces pmr1 mutant, an alpha 1,3-mannosyltransferase was active in microsomal extracts, but the alpha 1,3-Man epitope could not be identified on Western blots of cellular glycoproteins using sugar linkage-specific antibodies or lectins. The in vitro transferase assay is simple, rapid and accurate, and in the case of pmr1 suggests that in vivo either invertase is misrouted during secretion or the alpha 1,3-mannosyltransferase is mistargeted after its synthesis in this mutant.

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