GLYCOPROTEIN-BIOSYNTHESIS IN THE ALG3 SACCHAROMYCES-CEREVISIAE MUTANT.1. ROLE OF GLUCOSE IN THE INITIAL GLYCOSYLATION OF INVERTASE IN THE ENDOPLASMIC-RETICULUM

Oligosaccharides on invertase restricted to the endoplasmic reticulum (ER) in alg3,sec18 yeast at 37-degrees-C were found to be 20% wild typ e Man8GlcNAc and 80% ->2Man1alpha-->3(Man1alpha-->6)Man1beta-->4GlcNAc 2 (Verostek, M. F., Atkinson, P. H., and Trimble, R. B. (1991) J. Biol . Chem. 266, 5547-5551). These results suggested that alg3 was slightl y leaky, but did not address whether the oligosaccharide-lipid Man9Glc NAC2 and Man5GlcNAC2 precursors were glucosylated in alg3 yeast. There fore, an alg3,sec18,gls1 strain was constructed to delete the GLS1-enc oded glucosidase I responsible for trimming the terminal alpha1,2-link ed glucose from newly transferred Glc3ManxGlcNAC2 oligosaccharides. In vertase activity was overexpressed 5-10-fold on transforming this stra in with a multicopy plasmid (pRB58) carrying the SUC2 gene, and prepar ative amounts of the ER form of external invertase, derepressed and ac cumulated at 37-degrees-C, were purified. The N-linked glycans were re leased by sequential treatment with endo-beta-N-acetylglucosaminidase H (endo H) and peptide-N4-N-acetyl-beta-glucosaminyl asparagine amidas e. Oligosaccharide pools were sized separately on Bio-Gel P-4, which s howed that endo H released about 17% of the carbohydrate as Glc3Man8Gl cNAc, while peptide-N4-N-acetyl-beta-glucosaminyl asparagine amidase r eleased the remainder as Hex8GlcNAC2 and Man5GlcNAc2 in a 1:4 ratio. G lycan structures were assigned by 500-MHz two-dimensional DQF-COSY H-1 NMR spectroscopy, which revealed that the endo H-resistant Hex8GlcNAc 2 pool contained Glc3Man5GlcNAc2 and Man8GlcNAc2 in a 6:4 ratio, the l atter a different isomer from that formed by the ER alpha1,2-mannosida se (Byrd, J. C., Tarentino, A. L., Maley, F., Atkinson, P. H., and Tri mble, R. B. (1982) J. Biol. Chem. 257, 14657-14666). Recovery of Glc3M an8GlcNAc and not the ER form of Man8GlcNAc provided an internal contr ol indicating the absence of glucosidase I, which was confirmed by inc ubation of [H-3]Glc3[C-14]Man9GlcNAc with solubilized membranes from e ither alg3,sec18,gls1 or alg3,sec18,GLS1 strains. Chromatographic anal ysis of the products showed that [H03]Glc was removed only in the pres ence of the GLS1 gene product. Thus, the vast majority of the N-linked glycosylation in the ER of alg3 yeast (>75%) occurs by transfer of Ma n5GlcNAc2 without prior addition of the 3 glucoses normally found on t he lipid-linked precursor.