Abstract
Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) is an important cytosolic regulatory enzyme that plays a pivotal role in numerous physiological processes in plants, including seed development and germination. Previous studies demonstrated the occurrence of immunoreactive PEPC polypeptides of ~110kDa and 107kDa (p110 and p107, respectively) on immunoblots of clarified extracts of germinating sorghum (Sorghum bicolor) seeds. In order to establish the biochemical basis for this observation, a 460kDa PEPC heterotetramer composed of an equivalent ratio of p110 and p107 subunits was purified to near homogeneity from the germinated seeds. Mass spectrometry established that p110 and p107 are both encoded by the same plant-type PEPC gene (CP21), but that p107 was in vivo monoubiquitinated at Lys624 to form p110. This residue is absolutely conserved in vascular plant PEPCs and is proximal to a PEP-binding/catalytic domain. Anti-ubiquitin IgG immunodetected p110 but not p107, whereas incubation with a deubiquitinating enzyme (USP-2 core) efficiently converted p110 into p107, while relieving the enzyme’s feedback inhibition by l-malate. Partial PEPC monoubiquitination was also detected during sorghum seed development. It is apparent that monoubiquitination at Lys624 is opposed to phosphorylation at Ser7 in terms of regulating the catalytic activity of sorghum seed PEPC. PEPC monoubiquitination is hypothesized to fine-tune anaplerotic carbon flux according to the cell’s immediate physiological requirements for tricarboxylic acid cycle intermediates needed in support of biosynthesis and carbon–nitrogen interactions.
Highlights
Phosphoenolpyruvate (PEP) carboxylase (PEPC; EC role in the initial fixation of atmospheric CO2 during C4 and 4.1.1.31) catalyses the irreversible β-carboxylation of PEP Crassulacean acid metabolism photosynthesis
Preliminary immunoblot experiments corroborated an earlier report (Nhiri et al, 2000) that sorghum seed germination is accompanied by: (i) accumulation of comparable amounts of pre-existing approximate 107 kDa and inducible 110 kDa immunoreactive plant-type phosphoenolpyruvate carboxylase (PTPC) polypeptides (p107 and p110, respectively) (Fig. 1B); and (ii) in vivo phosphorylation of p107 at its conserved N-terminal seryl residue (Fig. 1C; anti-pSer13). Both subunits are intact since the anti-C19 and anti-N24 {antibodies raised against synthetic peptides corresponding to the C-terminal [(Y) 942-EDTLILTMKGIAAGMQNTG-960] and dephosphorylated N-terminal [4-ERHHSIDAQLRALAPGKVSEE24(YG)] ends, respectively, of C4-photosynthetic PTPC} both cross-reacted with p110 and p107 (Fig. 1C)
SDS– PAGE and immunoblotting indicated that the final preparation consisted of equivalent amounts of protein-staining or anti-castor oil seed (COS)-PTPC-immunoreactive p110 and p107 that respectively co-migrated with the subunits of purified Class-1 PEP carboxylase (PEPC) from germinated COS (Fig. 3A, B)
Summary
Phosphoenolpyruvate (PEP) carboxylase (PEPC; EC role in the initial fixation of atmospheric CO2 during C4 and 4.1.1.31) catalyses the irreversible β-carboxylation of PEP Crassulacean acid metabolism photosynthesis Non-photosynthetic functions, the anaplerotic replenishment of tricarboxylic acid (TCA) cycle intermediates consumed during biosynthesis and nitrogen assimilation (Echevarría and Vidal, 2003; O’Leary et al, 2011a). PTPC phosphorylation at its conserved N-terminal seryl residue has been widely studied, and is catalysed by a dedicated calcium-independent PTPC protein kinase (PPCK) This enhances allosteric activation of Class-1 PEPCs by hexose-phosphates while reducing inhibition by l-malate and l-aspartate (O’Leary et al, 2011a). Monoubiquitination of COS or harsh hakea Class-1 PEPC is inhibitory as it increased their Km(PEP) values while enhancing sensitivity to allosteric inhibitors These studies have provided a new paradigm for the post-translational control of non-photosynthetic Class-1 PEPCs, as well as the first examples of regulatory monoubiquitination of a metabolic enzyme in nature. A deubiquitinating enzyme, ataxin 3, is activated by monoubiquitination (Todi et al, 2009)
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