Abstract
O-linked N-acetylglucosamine modification (O-GlcNAcylation) is a nutrient-dependent protein post-translational modification (PTM), dynamically and reversibly driven by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) that catalyse the addition and the removal of the O-GlcNAc moieties to/from serine and threonine residues of target proteins respectively. Increasing evidence suggests involvement of O-GlcNAcylation in many biological processes, including transcription, signalling, neuronal development and mitochondrial function. The presence of a mitochondrial O-GlcNAc proteome and a mitochondrial OGT (mOGT) isoform has been reported. We explored the presence of mOGT in human cell lines and mouse tissues. Surprisingly, analysis of genomic sequences indicates that this isoform cannot be expressed in most of the species analysed, except some primates. In addition, we were not able to detect endogenous mOGT in a range of human cell lines. Knockdown experiments and Western blot analysis of all the predicted OGT isoforms suggested the expression of only a single OGT isoform. In agreement with this, we demonstrate that overexpression of the nucleocytoplasmic OGT (ncOGT) isoform leads to increased O-GlcNAcylation of mitochondrial proteins, suggesting that ncOGT is necessary and sufficient for the generation of the O-GlcNAc mitochondrial proteome.
Highlights
O-linked N-acetylglucosamine modification (O-GlcNAcylation) is a dynamic post-translational modification (PTM), characterized by the reversible covalent attachment of N-acetylglucosamine on serine and threonine residues of target proteins [1,2,3]
After the first report describing mitochondrial O-GlcNAc transferase (OGT) (mOGT) in 2003 [26], there have been no further reports on this isoform for more than a decade, whereas numerous studies have begun to uncover the functions of nucleocytoplasmic OGT (ncOGT)
Accumulating evidence suggests that OGlcNAcylation is an important regulator of many biological processes related to mitochondrial function [8]
Summary
O-linked N-acetylglucosamine modification (O-GlcNAcylation) is a dynamic post-translational modification (PTM), characterized by the reversible covalent attachment of N-acetylglucosamine on serine and threonine residues of target proteins [1,2,3]. Its primary role in many biological processes in eukaryotes has been established [4,5,6,7,8] many of the mechanisms underlying O-linked N-acetylglucosamine (OGlcNAc) function remain to be elucidated. Substrate specificity and regulatory mechanisms of OGT and OGA remain largely elusive, they are essential for stem cell viability and mouse embryonic development [14,15]. O-GlcNAcylation is a nutrient-dependent process and appears to be regulated by intracellular glucose availability [19,20]. 2–5 % of the intracellular glucose is metabolized through the hexosamine biosynthetic pathway, a metabolic process that utilizes one of the metabolites from glycolysis to produce UDP-GlcNAc, which is the high energy donor substrate of OGT [11,21]. Human OGT (hOGT) is a 116 kDa enzyme containing a C-terminal catalytic domain and
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