MANNOSYLTRANSFERASE ACTIVITIES IN MEMBRANES FROM VARIOUS YEAST STRAINS

In the yeast Golgi compartments, at least five, and potentially severa l additional mannosyltransferases are involved in elongating to 'manna n' the core Man(8)GlcNAc(2) oligosaccharide trimmed from Glc(3)Man(9)G lcNAc(2) in the endoplasmic reticulum, Structural studies on oligosacc harides from alg3 mutant yeast, which lack the four upper arm mannoses donated by Man-P-Dol (where Dol is dolichol), verified that the new a lpha 1,6-branch in endo H-resistant mannan in this strain is efficient ly initiated in vivo on the alpha 1,3-linked core residue of the lipid -oligosaccharide form of Man(5)GlcNAc(2) (Verostek et al., J. Biol. Ch em., 266, 5547-5551, 1991), This Man(5)GlcNAcGlcNAc[H-3]ol isomer (whe re GlcNAc[H-3]ol is N-acetylglucosamin [1-H-3] itol) was found to be a n excellent acceptor for a number of GDP-Man-dependent Golgi mannosylt ransferases in detergent-solubilized yeast membrane preparations: an a lpha 1,3-mannosyltransferase (Mnn1p), an alpha 1,6-mannosyltransferase (Och1p) and two alpha 1,2-mannosyltransferases (Mnt1p/Kre2p, ?) whose products were readily identified by H-1 NMR spectroscopy, The Man(6)G lcNAc-GlcNAc[H-3]ol isomers formed were easily defined by alpha 1,2-ma nnosidase sensitivity and either Bio-Gel P-4 gel filtration or AX-5 hi gh-performance liquid chromatography, In general, mannosyltransferases present in detergent-solubilized microsomes from most yeast strains m imicked the array of sugar linkages observed on their respective glyco proteins, However, in the case of the Saccharomyces pmr1 mutant, an al pha 1,3-mannosyltransferase was active in microsomal extracts, but the alpha 1,3-Man epitope could not be identified on Western blots of cel lular 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 a fter its synthesis in this mutant.