Phosphoenol pyruvate carboxylase (PEPC), a key enzyme responsible for replenishing components of the tricarboxylic acid (TCA) cycle in plants as well as in mammals, is responsible for fixing atmospheric carbon in addition to a number of non-photosynthetic reactions.1 One of these non-photosynthetic roles is anaplerotic, replenishing TCA cycle intermediates according to plant physiologic and metabolic needs. Strategies for controlling the anaplerotic actions of PEPC include post-translational phosphorylation and ubiquitination.
Ruiz-Ballestra et al.2 investigated mono-ubiquitination of plant-type PEPC, a post-translational modification (PTM) that fine-tunes this enzyme’s activity in other plant species,3 in germinating and developing Sorghum bicolor seeds. Using traditional immunologic and enzyme activity assays in addition to liquid chromatography coupled with mass spectrometry (LC-MS/MS), the researchers identified the site of the PTM and its effect on PEPC function.
Ruiz-Ballestra and co-workers grew sorghum plants, harvesting the seeds at varying stages of development and flash freezing them in liquid nitrogen. They also collected sorghum seeds at varying stages of germination in the same manner. Proteins extracted from the seeds were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the bands corresponding to PEPC identified immunologically. The peptides were analyzed using quadrupole time-of-flight mass spectrometry (QTOF-MS) or by LC-MS/MS using an Easy nLC Orbitrap Fusion Tribrid MS system (Thermo Scientific). The researchers compared the resulting MS/MS data with green plant protein sequences contained in the NCBI database.
The researchers confirmed that in sorghum seeds, plant-type PEPC exists as a heterotetramer comprising equal ratios of subunits p107 and p110. Using an anti-ubiquitin antibody, they showed that only the p110 subunit is ubiquitinated. Furthermore, upon incubation with de-ubiquitinating enzyme USP-2, the researchers showed that p110 arises from in vivo ubiquitination of p107. Using the QTOF proteomics data and Mascot analysis, Ruiz-Ballestra et al. confirmed that p110 and p107 are derived from the same plant-type PEPC (PTPC) gene, CP21. MS/MS data identified Lys624, a highly conserved residue in other vascular plants found close to a PEP-binding/catalytic domain, as the p110 ubiquitination site.
The investigators also assayed the activity of PEPC purified from the plant materials collected to determine the effect of increased levels of ubiquitination on the enzyme. They found that ubiquitinated PEPC (UB-PEPC) was more sensitive to L-malate inhibition. UB-PEPC catalyzed less carboxylation of PEP in the assay, but this was not due to any significant change in its binding ability to the substrate.
The researchers confirmed that p110 subunits’ levels were elevated in seed germination due to increased ubiquitination of p107, as shown previously. In addition, they found that levels of the p110 subunit are also raised in developing seeds. Therefore, as the seeds germinate and develop they become more sensitive to metabolic inhibition. Separate experiments using the same research materials showed that phosphorylation of PEPC, an alternative PTM that also modifies enzyme activity, can occur in tandem with ubiquitination and lead to the formation of different PEPC heterotetramers.
The researchers consider that mono-ubiquitination of PEPC in germinating and developing sorghum seeds is an in vivo strategy for regulating this enzyme’s activity in this species, as conserved in other plants.
1. O’Leary, B., et al. (2011) “The remarkable diversity of plant PEPC (phosphoenolpyruvate carboxylase): Recent insights into the physiological function and post-translational controls of nonphotosynthetic PEPCs,” Biochemical Journal, 436 (pp. 15–34).
2. Ruiz-Ballestra, I., et al. (2013) “In vivo monoubiquitination of anaplerotic phosphoenolpyruvate carboxylase occurs at Lys624 in germinating sorghum seeds,” Journal of Experimental Botany, doi:10.1093/jxb/ert386.
3. Shane, M.W., et al. (2013) “Reciprocal control of anaplerotic phosphoenolpyruvate carboxylase by in vivo monoubiquitination and phosphorylation in developing proteoid roots of phosphate-deficient Harsh Hakea,” Plant Physiology, 161 (pp. 1634–1644).
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