In vivo regulatory phosphorylation of the phosphoenolpyruvate carboxylase AtPPC1 in phosphate-starved Arabidopsis thaliana

PEPC [PEP(phosphoenolpyruvate) carboxylase] is a tightly controlled cytosolic enzyme Situated at a major branchpoint in plant metabolism. Accumulating evidence indicates important functions for PEPC and PPCK (PEPC kinase) in plant acclimation to nutritional P-i deprivation. However, little is known about the genetic origin or phosphorylation status of native PEPCs from -P-i (P-i-deficient) plants. The transfer of Arabidopsis suspension cells or seedlings to - P-i growth media resulted in: (i) the marked transcriptional upregulation of genes encoding the PEPC isoenzyme AtPPC1 (Arabidopsis thaliana PEPC1), and PPCK isoenzymes AtPPCK1 and AtPPCK2; (ii) >2-fold increases in PEPC specific activity and in the amount of an immunoreactive 107-kDa PEPC polypeptide (p107); and (iii) in vivo p107 phosphorylation as revealed by immunoblotting of clarified extracts with phosphosite-specific antibodies to Ser-11 (which could be reversed following P-i resupply). Approx. 1.3 mg of PEPC was purified 660-fold from -P-i suspension cells to apparent homogeneity with a specific activity of 22.3 units . mg(-1) of protein. Gel filtration, SDS/PAGE and immunoblotting demonstrated that purified PEPC exists as a 440-kDa homotetramer composed of identical p107 subunits. Sequencing of p 107 tryptic and Asp-N peptides by tandem MS established that this PEPC is encoded by AtPPC1. P-i-affinity PAGE coupled with immunoblotting indicated stoichiometric phosphorylation of the p107 Subunits of AtPPC1 at its conserved Ser-11 phosphorylation site. Phosphorylation activated AtPPC1 at pH 7.3 by lowering its K (PEP) and its sensitivity to inhibition by L-malate and L-aspartate, while enhancing activation by glucose 6-phosphate. Our results indicate that the simultaneous induction and in vivo phosphorylation activation of AtPPC1 contribute to the metabolic adaptations of - P-i Arabidopsis.