Two mitochondrial phosphatases, PP2c63 and Sal2, are required for posttranslational regulation of the TCA cycle in Arabidopsis

Abstract

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.

Establishment of a GC‐MS‐based 13C‐positional isotopomer approach suitable for investigating metabolic fluxes in plant primary metabolism

“…Glycolytic flux toward Glu synthesis is repressed by the mitochondrial TRX system in illuminated Arabidopsis leaves Several lines of evidence indicate that the metabolic fluxes toward the TCA cycle are inhibited in illuminated leaves, which is particularly due to the inhibition of key components of mitochondrial metabolism through transcriptional and post-translational mechanisms (Fonseca-Pereira et al, 2021;Møller et al, 2020;Nunes-Nesi et al, 2013;Tcherkez et al, 2012;Xu et al, 2021). Among these mechanisms, the light-dependent, phosphorylation-mediated inhibition of mitochondrial PDH is a clear barrier to the entrance of carbon to the TCA cycle (Tcherkez et al, 2005;Tovar-Méndez et al, 2003;Zhang et al, 2021). Our analysis reinforces this concept, and provides further insight into how light inhibition of the mitochondrial PDH restrict the metabolic fluxes from PEPc and glycolysis toward Glu synthesis.…”

Acetylation of conserved lysines fine‐tunes mitochondrial malate dehydrogenase activity in land plants

“…The site stoichiometry can be estimated by the assumption that in different conditions modified peptides should have their abundance changed inversely proportional to their non‐modified counterpart (Olsen et al., 2010). Here we selected light‐ and dark‐incubated samples based on the assumption that mitochondrial acetyl‐CoA levels change depending on the light‐controlled pyruvate dehydrogenase activity (Zhang et al., 2021), which in turn may affect mitochondrial protein acetylation (König et al., 2014a). In this analysis, relative ratios of 126 different protein acetylation sites were quantified in all three biological replicates and site occupancy levels were calculated by MaxQuant (Cox and Mann, 2008) (Table S5a).…”

“…Developmental and environmental factors are known to impact the abundance of PTMs to dynamically adjust metabolism to the actual needs of the cell and thus each experimental condition may only detect a subset of physiologically relevant acetylations in a given site stoichiometry. An extreme example is the light dependence of phosphorylation of metabolic enzymes, where no or full phosphorylation is detected depending on whether light is present or not (Kim et al., 2019; Zhang et al., 2021). Light‐ and acetate‐dependent regulation of protein lysine acetylation is also reported in Chlamydomonas , which also affected the plastidial MDH protein and resulted in higher acetylation levels under heterotrophic conditions (Füßl et al., 2021).…”

PEPc-mediated CO2assimilation provides carbons to gluconeogenesis and the TCA cycle in both dark-exposed and illuminated guard cells

“…13 C-NMR studies indicate that the synthesis of Glu in illuminated leaves strongly depends on stored compounds (Tcherkez et al, 2009;Gauthier et al, 2010;Abadie et al, 2017), which are presumably accumulated in the previous night period (Cheung et al, 2014). This is likely an alternative to overcome the light-inhibition of PDH (Tovar-Méndez et al, 2003;Zhang et al, 2021), an important source of carbons for the TCA cycle and associated pathways (Reid et al, 1977). The degradation of labelled pyruvate by PDH or PC would provide two and three labelled carbons into acetyl-CoA and OAA, respectively.…”