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 c hain elongation of N-linked glycoproteins show higher cell wall porosi ty than normal cells, and are hypersensitive to antibiotics with a lar ge molecular weight; such as neomycin and geneticin. Wild-type yeast c ells also showed enhanced sensitivity to neomycin in the presence of t unicamycin, an inhibitor of N-glycosylation, suggesting that the exten t of N-glycosylation may affect the sensitivity of yeast cells to drug s and that sensitivity to neomycin may be an effective method for scre ening for yeast mutants defective in N-glycosylation. Pursuing this lo gic, we isolated neomycin-sensitive yeast mutants and screened them fo r defects in N-glycosylation. The neomycin-sensitive, N-glycosylation- defective mutants fell into 15 complementation groups including allele s of the previously isolated temperature-sensitive nes mutants nes10, nes17, and nes25. Gene cloning revealed that NES10 was identical to SE C20, which is involved in ER-Golgi protein transport. NES17 was identi cal to ALG1, which encodes a beta 1,4-mannosyltransferase present in t he 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 sy nthesis. NES25 was identical to GUK1, which encodes a GMP kinase. Over expression of MSN17 increased the GDP-mannose level in a wild-type str ain by about threefold, and guk1 decreased the GDP-mannose level to on e-fourth, suggesting a close relationship between GTP metabolism and m annose outer chain elongation; the link is presumably provided by the process of GDP-mannose transport in the Golgi membranes.