Mitochondrial O-GlcNAc transferase (mOGT) regulates mitochondrial structure, function and survival in HeLa cells [Metabolism]

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 as siRNA-mediated knockdown of endogenous mOGT protected cells against toxicity mediated by rotenone, a complex I inhibitor. Conversely, reduced expression of both nucleocytoplasmic (nc) and mitochondrial (m) OGT isoforms is associated with increased mitochondrial respiration, and elevated glycolysis, suggesting that ncOGT is a negative regulator of cellular bioenergetics. Lastly, we determined that mOGT is likely 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 aconitate hydratase, mitochondrial. Our findings suggest that mOGT is catalytically active in vivo and supports mitochondrial structure, health and survival, whereas ncOGT predominantly regulates cellular bioenergetics.

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