title: In vivo monoubiquitination of anaplerotic phosphoenolpyruvate carboxylase occurs at Lys624 in germinating sorghum seeds.
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 ~110 kDa and 107 kDa (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 460 kDa 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.
authors.affiliation: Departamento de Biología Vegetal, Facultad de Biología, Universidad de Sevilla, Avda Reina Mercedes no. 6, 41012 Sevilla, Spain.
authors.forename: Isabel
authors.initials: I
authors.lastname: Ruiz-Ballesta
authors.forename: Ana-Belén
authors.initials: AB
authors.lastname: Feria
authors.forename: Hong
authors.initials: H
authors.lastname: Ni
authors.forename: Yi-Min
authors.initials: YM
authors.lastname: She
authors.forename: William Charles
authors.initials: WC
authors.lastname: Plaxton
authors.forename: Cristina
authors.initials: C
authors.lastname: Echevarría
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D018525:Germination; D015151:Immunoblotting; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D013058:Mass Spectrometry; D008970:Molecular Weight; D010730:Phosphoenolpyruvate Carboxylase / Q000302:isolation & purification / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits / Q000378:metabolism; D012639:Seeds / Q000201:enzymology* / Q000254:growth & development*; D045868:Sorghum / Q000201:enzymology* / Q000254:growth & development; D054875:Ubiquitination
chemical_list: D010940:Plant Proteins; D021122:Protein Subunits; D010730:Phosphoenolpyruvate Carboxylase; D008239:Lysine
keywords: Development; Sorghum bicolor.; germination; monoubiquitination; phosphoenolpyruvate carboxylase; post-translational modification; seeds