Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany; Center of Plant Systems Biology and Biotechnology, 4000 Plovdiv, Bulgaria.
Institute of Plant Biology and Biotechnology, University of Muenster, Schlossplatz 7, 48149 Muenster, Germany.
Mol Plant. 2021 Jul 5;14(7):1104-1118. doi: 10.1016/j.molp.2021.03.023. Epub 2021 Mar 31.
Protein phosphorylation is a well-established post-translational mechanism that regulates protein functions and metabolic pathways. It is known that several plant mitochondrial proteins are phosphorylated in a reversible manner. However, the identities of the protein kinases/phosphatases involved in this mechanism and their roles in the regulation of the tricarboxylic acid (TCA) cycle remain unclear. In this study, we isolated and characterized plants lacking two mitochondrially targeted phosphatases (Sal2 and PP2c63) along with pyruvate dehydrogenase kinase (PDK). Protein-protein interaction analysis, quantitative phosphoproteomics, and enzymatic analyses revealed that PDK specifically regulates pyruvate dehydrogenase complex (PDC), while PP2c63 nonspecifically regulates PDC. When recombinant PP2c63 and Sal2 proteins were added to mitochondria isolated from mutant plants, protein-protein interaction and enzymatic analyses showed that PP2c63 directly phosphorylates and modulates the activity of PDC, while Sal2 only indirectly affects TCA cycle enzymes. Characterization of steady-state metabolite levels and fluxes in the mutant lines further revealed that these phosphatases regulate flux through the TCA cycle, and that altered metabolism in the sal2 pp2c63 double mutant compromises plant growth. These results are discussed in the context of current models of the control of respiration in plants.
蛋白质磷酸化是一种成熟的翻译后修饰机制，可调节蛋白质功能和代谢途径。已知几种植物线粒体蛋白以可逆的方式发生磷酸化。然而，参与这种机制的蛋白激酶/磷酸酶的身份及其在三羧酸（TCA）循环调节中的作用尚不清楚。在这项研究中，我们分离并鉴定了两种定位于线粒体的磷酸酶（Sal2 和 PP2c63）以及丙酮酸脱氢酶激酶（PDK）缺失的植物。蛋白质-蛋白质相互作用分析、定量磷酸蛋白质组学和酶分析表明，PDK 特异性调节丙酮酸脱氢酶复合物（PDC），而 PP2c63 非特异性调节 PDC。当重组 PP2c63 和 Sal2 蛋白被添加到突变体植物分离的线粒体中时，蛋白质-蛋白质相互作用和酶分析表明，PP2c63 直接磷酸化并调节 PDC 的活性，而 Sal2 仅间接影响 TCA 循环酶。对突变体系中稳态代谢物水平和通量的表征进一步表明，这些磷酸酶调节 TCA 循环中的通量，而 sal2 pp2c63 双突变体中改变的代谢会影响植物生长。这些结果在植物呼吸控制的当前模型背景下进行了讨论。
