Identification of novel O-GlcNAc transferase substrates using yeast cells expressing OGT

J Gen Appl Microbiol. 2021 Apr 16;67(1):33-41. doi: 10.2323/jgam.2020.04.002. Epub 2020 Nov 21.


O-GlcNAc modification mediated by O-GlcNAc transferase (OGT) is a reversible protein modification in which O-GlcNAc moieties are attached to target proteins in the cytosol, nucleus, and mitochondria. O-GlcNAc moieties attached to proteins can be removed by O-GlcNAcase (OGA). The addition of an O-GlcNAc moiety can influence several aspects of protein function, and aberrant O-GlcNAc modification is linked to a number of diseases. While OGT and OGA are conserved across eukaryotic cells, yeasts lack these enzymes. Previously, we reported that protein O-GlcNAc modification occurred in the budding yeast Saccharomyces cerevisiae when OGT was ectopically expressed. Because yeast cells lack OGA, O-GlcNAc moieties are stably attached to target proteins. Thus, the yeast system may be useful for finding novel OST substrates. By proteomic analysis, we identified 468 O-GlcNAcylated proteins in yeast cells expressing human OGT. Among these proteins, 13 have human orthologues that show more than 30% identity to their corresponding yeast orthologue, and possible glycosylation residues are conserved in these human orthologues. In addition, the orthologues have not been reported as substrates of OGT. We verified that some of these human orthologues are O-GlcNAcylated in cultured human cells. These proteins include an ubiquitin-conjugating enzyme, UBE2D1, and an eRF3-similar protein, HBS1L. Thus, the yeast system would be useful to find previously unknown O-GlcNAcylated proteins and regulatory mechanisms.

Keywords: O-GlcNAc; O-GlcNAc transferase; branched-chain amino acid aminotransferase; ubiquitin-conjugating enzyme; yeast.

MeSH terms

  • Gene Expression Regulation, Fungal
  • Glycosylation
  • HEK293 Cells
  • Humans
  • N-Acetylglucosaminyltransferases / genetics*
  • N-Acetylglucosaminyltransferases / isolation & purification*
  • N-Acetylglucosaminyltransferases / metabolism*
  • Protein Processing, Post-Translational
  • Proteomics
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae / genetics*


  • N-Acetylglucosaminyltransferases
  • O-GlcNAc transferase
  • OGT protein, human