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Decreased O‐glycosylation and drug resistance of Saccharomyces cerevisiae vrg4 mutant cells
Author(s) -
Ellerton Sharon Speiser,
Litewka Anna Justyna,
Jue Chong Kwong
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.2_supplement.278
Subject(s) - spheroplast , mutant , lysis , mannose , saccharomyces cerevisiae , biochemistry , mannan , microbiology and biotechnology , cell wall , glycosylation , chemistry , chitinase , tunicamycin , biology , enzyme , yeast , escherichia coli , polysaccharide , unfolded protein response , gene
In S. cerevisiae the VRG4 protein participates in mannan biosynthesis. We studied mannan synthesis in the mutant vrg4 by measuring the degree of protein O ‐glycosylation. The O ‐glycosylated protein, chitinase, was isolated and run on SDS gels. The mutant's chitinase band migrated faster than that of wild type (WT) indicating a decreased level of mannose incorporation. We then studied the effect of abnormal cell wall mannosylation on cell wall integrity by measuring the rate of spheroplast formation and subsequent lyses in the presence of Zymolyase. Vrg4 was highly sensitive to Zymolyase, causing rapid cell lyses. After a 3 minute incubation with Zymolyase the mutant exhibited a 24% decrease in optical density at 600nm as compared with WT where no change was observed. After 9 minutes, there was a 74% decrease in optical density for the mutant as compared with 2% decrease for the WT. Sensitivity to Congo red was studied using 10‐fold dilution series of WT and vrg4 cells (5 × 0 5 to 5 × 10 1 ) grown on agar plates containing Congo red. The WT exhibited a very mild inhibition of growth at a density of 5 × 10 2 cells while vrg4 cells exhibited inhibition of growth at a cell density of 5 × 10 3 with no growth of colonies at lower concentrations. These results demonstrate the importance of VRG4 in O ‐glycosylation and maintaining the integrity of the cell wall. This work was supported by a PSC‐CUNY grant that was awarded to Chong Jue.