Abstract
Phosphoenolpyruvate carboxylase (PEPC) is a cytosolic, homotetrameric enzyme that serves a variety of functions in plants, acting as the primary form of CO2 fixation in the C4 photosynthesis pathway (C4-PEPC). In a previous work we have shown that C4-PEPC bind anionic phospholipids, resulting in PEPC inactivation. Also, we showed that PEPC can associate with membranes and to be partially proteolyzed. However, the mechanism controlling this remains unknown. Using semi purified-PEPC from sorghum leaf and a panel of PEPC-specific antibodies, we analyzed the conformational changes in PEPC induced by anionic phospholipids to cause the inactivation of the enzyme. Conformational changes observed involved the exposure of the C-terminus of PEPC from the native, active enzyme conformation. Investigation of the protease activity associated with PEPC demonstrated that cysteine proteases co-purify with the enzyme, with protease-specific substrates revealing cathepsin B and L as the major protease species present. The anionic phospholipid-induced C-terminal exposed conformation of PEPC appeared highly sensitive to the identified cathepsin protease activity and showed initial proteolysis of the enzyme beginning at the N-terminus. Taken together, these data provide the first evidence that anionic phospholipids promote not only the inactivation of the PEPC enzyme, but also its proteolysis.
Highlights
C4-phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) catalyzes the first carboxylation step in C4 photosynthesis
Sorghum C4-PEPC is a phosphatidic acid (PA)-binding protein that is inhibited in the presence of PA and other anionic phospholipids
Following incubation of sp-PEPC with PA, immunoprecipitation of PEPC was observed (Figures 1A,B, lanes PA). Both the anionic phospholipids PI and LPA have been previously described as inhibitors of PEPC activity (Monreal et al, 2010), and treatment of sp-PEPC with either of these anionic phospholipids, in this study, resulted in exposure of the PEPC C-terminus to a similar extent as for PA (Figure 1A, PI and LPA; Figure 1B, LPA)
Summary
C4-phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) catalyzes the first carboxylation step in C4 photosynthesis. Of the PTPCs, one family member, SbPEPC1, is an example of a C4 photosynthetic PEPC, playing a functional role in C4 and Crassulacean acid metabolism (CAM)-type photosynthesis, while the remaining four homologs, SbPEPC2-5, are C3 PEPCs, that perform alternative functions in plants (O’Leary et al, 2011). In the susbtrate-binding center, Ala774 (Flaveria numbering) mediates C3 specificity, while Ser774 determines the increased kinetic efficiency of C4 PEPC (Paulus et al, 2013). Malate binding in the inhibitory site, is controlled by Arg884 in the C3 enzyme while, the increased tolerance of C4-PEPC to the malate inhibitor is mediated by Gly884 (Paulus et al, 2013). C3-type plant PEPCs have been shown to be regulated by monoubiquitination (Uhrig et al, 2008; Shane et al, 2013; Ruiz-Ballesta et al, 2014, 2016), though this has not been demonstrated for the photosynthetic C4-PEPC to date (unpublished results)
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