Sacoman Juliana L, Dagda Raul Y, Burnham-Marusich Amanda R, Dagda Ruben K, Berninsone Patricia M
From the Department of Biology, University of Nevada, Reno, Nevada 89557 and.
the Departments of Pharmacology and.
J Biol Chem. 2017 Mar 17;292(11):4499-4518. doi: 10.1074/jbc.M116.726752. Epub 2017 Jan 18.
-Linked -acetylglucosamine transferase (OGT) catalyzes -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 and supports mitochondrial structure, health, and survival, whereas ncOGT predominantly regulates cellular bioenergetics.
O-连接的N-乙酰葡糖胺转移酶（OGT）催化靶蛋白的O-GlcNAc化，并调节众多生物学过程。OGT由一个产生核质和线粒体异构体的单一基因编码。迄今为止，OGT的线粒体异构体（mOGT）的作用仍 largely unknown。使用高通量蛋白质组学，我们鉴定了84种候选线粒体糖蛋白，其中44种是新的。值得注意的是，鉴定出的两种候选糖蛋白（细胞色素氧化酶2（COX2）和NADH：泛醌氧化还原酶核心亚基4（MT-ND4））由线粒体DNA编码。在HeLa细胞中使用小干扰RNA（siRNA），我们发现降低内源性mOGT表达会导致线粒体结构和功能的改变，包括依赖动力相关蛋白1（Drp1）的线粒体碎片化、线粒体膜电位降低以及在没有线粒体活性氧的情况下线粒体含量的显著损失。这些缺陷与每个线粒体氧化磷酸化的代偿性增加有关。mOGT对细胞存活也至关重要；siRNA介导的内源性mOGT敲低保护细胞免受鱼藤酮（一种复合物I抑制剂）介导的毒性。相反，核质（ncOGT）和线粒体（mOGT）OGT异构体的表达降低与线粒体呼吸增加和糖酵解升高有关，表明ncOGT是细胞生物能量学的负调节因子。最后，我们确定mOGT可能参与一组受限的线粒体靶标的糖基化。我们鉴定出四种与线粒体生物发生和代谢调节有关的蛋白质作为mOGT的候选底物，包括富含亮氨酸的含PPR蛋白和线粒体乌头酸水合酶。我们的研究结果表明，mOGT具有催化活性，并支持线粒体结构、健康和存活，而ncOGT主要调节细胞生物能量学。
