Identification of non-specific phosphatases in TCA cycle regulation

Protein phosphorylation is a well-established post-translational mechanism that regulates protein functions and metabolic pathways. It has been shown that several plant mitochondrial proteins are phosphorylated in a reversible manner. However, the identity of the phosphatases/kinases involved in this mechanism and their role in the regulation of the TCA cycle remains unclear. Here, we isolated and characterized plants lacking two mitochondrially targeted phosphatases (Sal2 and PP2c63) alongside pyruvate dehydrogenase kinase (PDK). Protein-protein interaction, quantitative phosphoproteomics and enzymatic analyses revealed that PDK specifically regulates pyruvate dehydrogenase (PDH), whilst Sal2 and PP2c63 regulate PDHand fumarase. Alongside with the recombinant protein complemented the purified mitochondria, the PP2c63 directly regulate the PDH and fumarase while the Sal2 indirectly regulated. Characterization of steady-state metabolite levels and fluxes in the corresponding mutants revealed that these phosphatases regulate the fluxes through the TCA cycle, with altered metabolism compromising growth of the sal2/pp2c63 double. The combined data are collectively discussed in the context of current models of the control of respiration in plants.