A defect in GTP synthesis affects mannose outer chain elongation in Saccharomyces cerevisiae.
We have found that yeast mutants that are defective in mannose outer chain elongation of N-linked glycoproteins show higher cell wall porosity than normal cells, and are hypersensitive to antibiotics with a large molecular weight; such as neomycin and geneticin. Wild-type yeast cells also showed enhanced sensitivity to neomycin in the presence of tunicamycin, an inhibitor of N-glycosylation, suggesting that the extent of N-glycosylation may affect the sensitivity of yeast cells to drugs and that sensitivity to neomycin may be an effective method for screening for yeast mutants defective in N-glycosylation. Pursuing this logic, we isolated neomycin-sensitive yeast mutants and screened them for defects in N-glycosylation. The neomycin-sensitive, N-glycosylation-defective mutants fell into 15 complementation groups including alleles of the previously isolated temperature-sensitive nes mutants nes10, nes17, and nes25. Gene cloning revealed that NES10 was identical to SEC20, which is involved in ER-Golgi protein transport, NES17 was identical to ALG1, which encodes a beta-1,4-mannosyltransferase present in the ER, MSN17, a multicopy suppressor of nes17/alg1, was also isolated and found to be an allele of PSA1, which is involved in GDP-mannose synthesis, NES25 was identical to GUK1, which encodes a GMP kinase. Overexpression of MSN17 increased the GDP-mannose level in a wild-type strain by about threefold, and guk1 decreased the GDP-mannose level to one-fourth, suggesting a close relationship between GTP metabolism and mannose outer chain elongation; the link is presumably provided by the process of GDP-mannose transport in the Golgi membranes.
Shimma
Y
Y
Molecular Biology Department, National Institute of Bioscience and Human Technology, Ibaraki, Japan.
Nishikawa
A
A
bin Kassim
B
B
Eto
A
A
Jigami
Y
Y
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002473:Cell Wall; D006031:Glycosylation; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000096:biosynthesis* / Q000378:metabolism; D051528:Guanylate Kinases; D008358:Mannose / Q000378:metabolism*; D008364:Mannosyltransferases / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009355:Neomycin / Q000494:pharmacology; D009703:Nucleoside-Phosphate Kinase / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
D000900:Anti-Bacterial Agents; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D008364:Mannosyltransferases; C035777:chitobiosyldiphosphodolichol beta-mannosyltransferase; D009703:Nucleoside-Phosphate Kinase; D051528:Guanylate Kinases; D009355:Neomycin; D008358:Mannose