Disruption of the processing alpha-mannosidase gene does not prevent outer chain synthesis in Saccharomyces cerevisiae.
Processing of N-linked oligosaccharides in Saccharomyces cerevisiae begins with the removal of glucose and mannose residues from Glc3Man9GlcNAc2 to form a single isomer of Man8GlcNAc2. The importance of mannose removal for subsequent outer chain synthesis was examined in strains of S. cerevisiae disrupted in the MNS1 gene encoding a specific alpha 1,2-mannosidase responsible for Man8GlcNAc2 synthesis [Camirand, Heysen, Grondin and Herscovics (1991) J. Biol. Chem. 266, 15120-15127]. Both MNS1 transcripts of 1.85 kb and 1.7 kb were not observed in Northern blots of mns1 cells (i.e. cells containing the disrupted gene). Analysis on Bio-Gel P-6 of endo-beta-N-acetylglucosaminidase-H-sensitive oligosaccharides following a 10 min pulse with [2-3H]mannose revealed similar amounts of labelled outer chains excluded from the gel in both control and mns1 cells. H.p.l.c. of the included oligosaccharides showed that a Man9GlcNAc, rather than a Man8GlcNAc, intermediate was formed in mns1 cells. Analysis of [3H]mannose-labelled core oligosaccharides from immunoprecipitated CPY and invertase by h.p.l.c. showed a similar size distribution in mns1 and control cells. Invertase immunoprecipitated from [35S]methionine-labelled mns1 cells was highly glycosylated, but migrated slightly faster than that from control cells on denaturing PAGE, indicating a small difference in glycosylation. A similar difference in mobility was observed for invertase activity stain following non-denaturing gel electrophoresis. It is concluded that the alpha-mannosidase encoded by MNS1 is the only enzyme responsible for mannose removal in vivo, and that this processing step is not essential for outer chain synthesis.
Puccia
R
R
McGill Cancer Centre, McGill University, Montreal, Quebec, Canada.
Grondin
B
B
Herscovics
A
A
D015152:Blotting, Northern; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005800:Genes, Fungal; D006023:Glycoproteins / Q000096:biosynthesis; D006026:Glycoside Hydrolases / Q000032:analysis / Q000378:metabolism; D006031:Glycosylation; D006596:Hexosaminidases / Q000378:metabolism; D007163:Immunosorbent Techniques; D008358:Mannose / Q000378:metabolism; D008361:Mannosidases / Q000096:biosynthesis / Q000235:genetics*; D009154:Mutation; D009844:Oligosaccharides / Q000032:analysis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D014316:Tritium; D043323:alpha-Mannosidase; D043324:beta-Fructofuranosidase
D006023:Glycoproteins; D009844:Oligosaccharides; D014316:Tritium; D006026:Glycoside Hydrolases; D006596:Hexosaminidases; D008361:Mannosidases; D043323:alpha-Mannosidase; D043324:beta-Fructofuranosidase; D008358:Mannose