Mutation in alg10 gene supresses the growth defect of Schizosaccharomyces pombe glucosidase I mutants

GALLO, GL; GARCIA, MD; FARAONI, EY; HERRERA AGUILAR, N; PARODI AJ; D'ALESSIO, C.

Buenos Aires

Simposio; 1er Simposio Argentino de Glicobiología (GlycoAR 2014); 2014

Glucosidase I is responsible of the initial step in N-glycan processing as it removes the outermost glucose from the Glc3Man9GlcNAc2 transferred upon protein N-glycosylation. Surprisingly, the S. pombe genome-wide deletion mutant project reported that, contrary to Saccharomyces cerevisiae, disruption of Glucosidase I in S. pombe is lethal. We induced meiosis and sporulation in a heterozygous Dgls1/+ strain and obtained two viable haploid Dgls1 strains: one with a severe but not lethal growth phenotype (10A) and another with a less severe growth defect (10D). We analyzed both the protein-linked and the lipid-linked oligosaccharides in each strain. While 10A strain produced only Glc3Man9GlcNAc2 glycans linked to proteins, 10D strain produced a mixture of Glc3Man9GlcNAc2 and Man9GlcNAc2. The lipid-linked oligosaccharide pattern of strain 10A showed that the most abundant species formed was Glc3Man9GlcNAc2, while in 10D strain it was Glc2Man9GlcNAc2 and a small amount of Glc3Man9GlcNAc2, indicating a very reduced alg10 gene activity and that a mutation in alg10 gene may be responsible of the suppression of the growth defect in 10D strain. We constructed a Dgls1/Dalg10 double knock out mutant. As expected, this strain showed a normal growth rate. We propose that the accumulation of glycoproteins bearing three glucoses is responsible of the growth defect observed in Glucosidase I minus mutants