title: MitochondrialO-GlcNAc Transferase (mOGT) Regulates Mitochondrial Structure, Function, and Survival in HeLa Cells.
abstract: O-LinkedN-acetylglucosamine transferase (OGT) catalyzesO-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 activein vivoand supports mitochondrial structure, health, and survival, whereas ncOGT predominantly regulates cellular bioenergetics.
authors.affiliation: From the Department of Biology, University of Nevada, Reno, Nevada 89557 and.
authors.forename: Juliana L
authors.initials: JL
authors.lastname: Sacoman
authors.affiliation: the Departments of Pharmacology and.
authors.forename: Raul Y
authors.initials: RY
authors.lastname: Dagda
authors.affiliation: Microbiology and Immunology, University of Nevada, Reno School of Medicine, Reno, Nevada 89557.
authors.forename: Amanda R
authors.initials: AR
authors.lastname: Burnham-Marusich
authors.affiliation: the Departments of Pharmacology and rdagda@medicine.nevada.edu.
authors.forename: Ruben K
authors.initials: RK
authors.lastname: Dagda
authors.affiliation: From the Department of Biology, University of Nevada, Reno, Nevada 89557 and berninsone@unr.edu.
authors.forename: Patricia M
authors.initials: PM
authors.lastname: Berninsone
mesh_terms: D002470:Cell Survival; D006019:Glycolysis; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D053078:Membrane Potential, Mitochondrial; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D024101:Mitochondrial Proteins; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D017351:N-Acetylglucosaminyltransferases; C449880:O-GlcNAc transferase
keywords: O-GlcNAcylation; energy metabolism; glycosylation; mitochondria; mitochondrial metabolism