Isolation of protein glycosylation mutants in the fission yeast Schizosaccharomyces pombe.
We have isolated mutants in the fission yeast Schizosaccharomyces pombe that are defective in protein glycosylation. A collection of osmotically sensitive mutants was prepared and screened for glycosylation defects using lectin staining as an assay. Mutants singly defective in four glycoprotein synthesis genes (gps1-4) were isolated, all of which bind less galactose-specific lectin. Acid phosphatase and other glycoproteins from the gps mutants have increased electrophoretic mobility, suggesting that these mutants make glycans of reduced size. N-linked glycan analysis revealed that terminal oligosaccharide modification is defective in the gps1 and gps2 mutants. Both mutants synthesize the Man9GlcNAc2 core glycan but have reduced amounts of larger structures. Modified core glycans from gps1 cells have normal amounts of galactose (Gal) residues, but reduced amounts of Man, consistent with a defect in a Golgi mannosyltransferase in this mutant. In contrast, N-linked oligosaccharides from gps2 mutants have much less Gal than wild type, because of reduced levels of the Gal donor, UDP-Gal. This reduction is caused by decreased activity of UDP-glucose 4-epimerase, which synthesizes UDP-Gal. Neither the gps1 or gps2 mutations are lethal, although the cells grow at reduced rates. These findings suggest that S. pombe cells can survive with incompletely glycosylated cell wall glycoproteins. In particular, these results suggest that Gal, which comprises approximately 30% by weight of cell wall glycoprotein glycans, is not crucial for cell growth or survival.
Huang
K M
KM
Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4935, USA.
Snider
M D
MD
D000135:Acid Phosphatase / Q000032:analysis; D002473:Cell Wall / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism*; D005690:Galactose / Q000378:metabolism; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D006031:Glycosylation; D037102:Lectins; D008351:Mannans / Q000096:biosynthesis; D008562:Membrane Glycoproteins / Q000378:metabolism*; D009154:Mutation; D009844:Oligosaccharides / Q000378:metabolism; D009997:Osmotic Pressure; D037121:Plant Lectins; D011134:Polysaccharides / Q000096:biosynthesis; D012568:Schizosaccharomyces / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D014534:UDPglucose 4-Epimerase / Q000378:metabolism; D014531:Uridine Diphosphate Galactose / Q000096:biosynthesis; D014532:Uridine Diphosphate Glucose / Q000096:biosynthesis
D005656:Fungal Proteins; C045475:Griffonia simplicifolia lectins; D037102:Lectins; D008351:Mannans; D008562:Membrane Glycoproteins; D009844:Oligosaccharides; D037121:Plant Lectins; D011134:Polysaccharides; D014531:Uridine Diphosphate Galactose; C057328:mannosyl(9)-N-acetylglucosamine; D000135:Acid Phosphatase; D014534:UDPglucose 4-Epimerase; D014532:Uridine Diphosphate Glucose; D005690:Galactose