Hideki Nakanishi1, Feng Li2, Baoxian Han2, Seisuke Arai3, Xiao-Dong Gao4. 1. Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China. Electronic address: hideki@jiangnan.edu.cn. 2. Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China. 3. Department of Cell Science, Institute of Biomedical Science, Fukushima Medical University School of Medicine, Fukushima, Japan. 4. Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China. Electronic address: xdgao@jiangnan.edu.cn.
Abstract
BACKGROUND: O-GlcNAcylation is a reversible protein post-translational modification, where O-GlcNAc moiety is attached to nucleocytoplasmic protein by O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA). Although O-GlcNAc modification widely occurs in eukaryotic cells, the budding yeast Saccharomyces cerevisiae notably lacks this protein modification and the genes for the GlcNAc transferase and hydrolase. METHODS: Human OGT isoforms and OGA were ectopically expressed in S. cerevisiae, and the effects of their expressions on yeast growth and O-GlcNAc modification levels were assessed. RESULTS: Expression of sOGT, in S. cerevisiae catalyzes the O-GlcNAc modification of proteins in vivo; conversely, the expression of OGA mediates the hydrolysis of these sugars. sOGT expression causes a severe growth defect in yeast cells, which is remediated by the co-expression of OGA. The direct analysis of yeast proteins demonstrates protein O-GlcNAcylation is dependent on sOGT expression; conversely, the hydrolysis of these sugar modifications is induced by co-expression of OGA. Protein O-GlcNAcylation and the growth defects of yeast cells are caused by the O-GlcNAc transferase activity because catalytically inactive sOGT does not exhibit toxicity in yeast cells. Expression of another OGT isoform, ncOGT, also results in a growth defect in yeast cells. However, its toxicity is largely attributed to the TPR domain rather than the O-GlcNAc transferase activity. CONCLUSIONS: O-GlcNAc cycling can occur in yeast cells, and OGT and OGA activities can be monitored via yeast growth. GENERAL SIGNIFICANCE: Yeast cells may be used to assess OGT and OGA.
BACKGROUND: O-GlcNAcylation is a reversible protein post-translational modification, where O-GlcNAc moiety is attached to nucleocytoplasmic protein by O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA). Although O-GlcNAc modification widely occurs in eukaryotic cells, the budding yeastSaccharomyces cerevisiae notably lacks this protein modification and the genes for the GlcNAc transferase and hydrolase. METHODS:HumanOGT isoforms and OGA were ectopically expressed in S. cerevisiae, and the effects of their expressions on yeast growth and O-GlcNAc modification levels were assessed. RESULTS: Expression of sOGT, in S. cerevisiae catalyzes the O-GlcNAc modification of proteins in vivo; conversely, the expression of OGA mediates the hydrolysis of these sugars. sOGT expression causes a severe growth defect in yeast cells, which is remediated by the co-expression of OGA. The direct analysis of yeast proteins demonstrates protein O-GlcNAcylation is dependent on sOGT expression; conversely, the hydrolysis of these sugar modifications is induced by co-expression of OGA. Protein O-GlcNAcylation and the growth defects of yeast cells are caused by the O-GlcNAc transferase activity because catalytically inactive sOGT does not exhibit toxicity in yeast cells. Expression of another OGT isoform, ncOGT, also results in a growth defect in yeast cells. However, its toxicity is largely attributed to the TPR domain rather than the O-GlcNAc transferase activity. CONCLUSIONS:O-GlcNAc cycling can occur in yeast cells, and OGT and OGA activities can be monitored via yeast growth. GENERAL SIGNIFICANCE: Yeast cells may be used to assess OGT and OGA.