Abstract
O-Linked N-acetylglucosamine transferase (OGT) catalyzes O-GlcNAcylation of target proteins and regulates numerous biological processes. OGT is encoded by a single gene that yields nucleocytosolic and mitochondrial isoforms. To date, the role of the mitochondrial isoform of OGT (mOGT) remains largely unknown. Using high throughput proteomics, we identified 84 candidate mitochondrial glycoproteins, of which 44 are novel. Notably, two of the candidate glycoproteins identified (cytochrome oxidase 2 (COX2) and NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4)) are encoded by mitochondrial DNA. Using siRNA in HeLa cells, we found that reducing endogenous mOGT expression leads to alterations in mitochondrial structure and function, including Drp1-dependent mitochondrial fragmentation, reduction in mitochondrial membrane potential, and a significant loss of mitochondrial content in the absence of mitochondrial ROS. These defects are associated with a compensatory increase in oxidative phosphorylation per mitochondrion. mOGT is also critical for cell survival; siRNA-mediated knockdown of endogenous mOGT protected cells against toxicity mediated by rotenone, a complex I inhibitor. Conversely, reduced expression of both nucleocytoplasmic (ncOGT) and mitochondrial (mOGT) OGT isoforms is associated with increased mitochondrial respiration and elevated glycolysis, suggesting that ncOGT is a negative regulator of cellular bioenergetics. Last, we determined that mOGT is probably involved in the glycosylation of a restricted set of mitochondrial targets. We identified four proteins implicated in mitochondrial biogenesis and metabolism regulation as candidate substrates of mOGT, including leucine-rich PPR-containing protein and mitochondrial aconitate hydratase. Our findings suggest that mOGT is catalytically active in vivo and supports mitochondrial structure, health, and survival, whereas ncOGT predominantly regulates cellular bioenergetics.
Highlights
O-Linked N-acetylglucosamine transferase (OGT) catalyzes O-GlcNAcylation of target proteins and regulates numerous biological processes
MOGT Glycosylates a Restricted Set of Mitochondrial Proteins—Because our overall data indicated that reducing the endogenous levels of mitochondrial isoform of OGT (mOGT) significantly affects mitochondrial structure and some aspects of mitochondrial function, we investigated whether alterations in mitochondrial structure and function caused by a reduction of endogenous levels of mOGT are associated with altered mOGT-mediated O-GlcNAcylation of mitochondrial proteins
A role of mOGT in regulating mitochondrial metabolism and function has been proposed [38], but the function of mOGT was investigated in the context of overexpression [26]
Summary
O-GlcNAcylation, O-linked N-acetylglucosamine glycosylation; OGT, O-GlcNAc transferase; mOGT, mitochondrial OGT; ncOGT, nucleocytoplasmic OGT; NT, non-targeting; OGA, OGlcNAcase; qPCR, quantitative PCR; ANOVA, analysis of variance; DIGE, differential in-gel electrophoresis; OCR, oxygen consumption rate; WGA, wheat germ agglutinin; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; TMRM, tetramethylrhodamine, methylester, perchlorate. Overexpression of the nucleocytoplasmic OGT isoform (ncOGT) decreases mitochondrial respiration and leads to abnormal formation of mitochondrial cristae [25] These contrasting roles underscore the critical need to close the gap in our knowledge regarding the molecular mechanisms that govern O-GlcNAcylation of mitochondrial proteins. Overexpression of mOGT triggers apoptosis, a phenotype that is dependent on the mitochondrial localization of mOGT and its catalytic activity [26] This observation suggests that deregulated mitochondrial O-GlcNAcylation plays a role in programmed cell death signaling. We identified four candidate glycoproteins that may exhibit reduced glycosylation when endogenous mOGT levels are decreased by siRNA, suggesting that mOGT is catalytically active in vivo and that glycosylation of these proteins may be part of the mechanism by which mOGT regulates mitochondrial physiology and function
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