Phosphoenolpyruvate carboxylase from C4 leaves is selectively targeted for inhibition by anionic phospholipids.

Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) is an enzyme playing a crucial role in photosynthesis of C4 plants. Here, we identify anionic phospholipids as novel regulators that inhibit C4 PEPC activity and provide evidence that the enzyme partially localizes to membranes. PEPC catalyzes the b-carboxylation of phosphoenolpyruvate (PEP) in a reaction that yields oxaloacetate and inorganic phosphate. In C4 plants, it notably performs the initial fixation of atmospheric CO2 in photosynthesis, while it also has an anaplerotic function in coordinating carbon and nitrogen metabolism in all plants (Chollet et al., 1996; Vidal and Chollet, 1997). The PEPC protein is subject to distinct but interrelated mechanisms of posttranslational regulation by allosteric positive (e.g. Glc-6-P, triose-P) or negative (e.g. L-malate, Asp) effectors, as well as phosphorylation of the protein at its N-terminal domain (Nimmo, 2003). Previously, we have identified C3 PEPC isoforms as phosphatidic acid (PA)-binding proteins from tomato (Solanum lycopersicum) and Arabidopsis (Arabidopsis thaliana) suspension-cultured cells in a proteomics screen (Testerink et al., 2004). Phospholipids can affect both localization and activity of a diverse range of proteins, including protein kinases and phosphatases (Testerink and Munnik, 2005; Hurley, 2006; Lemmon, 2008; Munnik and Testerink, 2009; Stahelin, 2009), but also directly regulate metabolic enzymes, such as Escherichia coli pyruvate oxidase (Neumann et al., 2008), mammalian CTP:phosphocholine cytidyltransferase (Johnson et al., 2003; Cornell and Taneva, 2006; Taneva et al., 2008), and wheat (Triticum aestivum) phosphoethanolamine N-methyltransferase (Jost et al., 2009). Using PA-affinity beads, we show here that C4-type PEPC also binds PA.Moreover, we found that C4 PEPC activity, unlike its C3 counterpart, was inhibited by addition of PA. Other anionic phospholipids, but not neutral, zwitterionic, or positively charged lipids, were also able to block PEPC activity. Western analysis of crude biochemical fractions of sorghum (Sorghum bicolor) leaf extracts revealed that although most PEPC was present in the soluble fraction, a subpool was membrane associated. A possible physiological role for PEPC recruitment to membranes is discussed.