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.

Keywords: Development; Sorghum bicolor.; germination; monoubiquitination; phosphoenolpyruvate carboxylase; post-translational modification; seeds.

Fig. 1.

Immunocharacterization of p110 and p107 PEPC subunits during sorghum seed germination. (A) Stages of seed germination; DS, dry seed. (B) Seed extracts were subjected to SDS–PAGE (8% gel, 40 μg of protein), electroblotted onto a PVDF membrane, and probed with anti-C₄ PEPC. (C) Immunoblots of 48h post-imbibition extracts were probed with anti-N24, anti-pSer13, and anti-C-19. (D) Monoubiquitination of p110. Clarified extracts from seeds imbibed in water for 48h were incubated in the presence or absence of 1 μM USP-2 core (Abcam) for 1h at 30 ºC, and subjected to immunoblot analysis using anti-C₄ PEPC; 50 μg protein lane^–1.

Fig. 2.

Co-elution of PEPC activity with 110kDa and 107kDa PEPC polypeptides (p110 and p107, respectively) during Superdex-200 HR 16/60 gel-filtration FPLC of PEPC from germinating sorghum seeds. Aliquots (10 μl each) from various fractions were subjected to SDS–PAGE and immunoblot analysis using anti-COS PTPC. V₀ denotes the column’s void volume.

Fig. 3.

SDS–PAGE and immunoblot analysis of various fractions obtained during the purification of PEPC from 48h germinated sorghum seeds. (A) SDS–PAGE was followed by protein staining with Coomassie Blue R-250 (CBB-250). (B) Immunoblot analysis was performed using anti-COS PTPC. Lane 1, 2.5 μg (A) or 50ng (B) of purified monoubiquitinated Class-1 PEPC (RcPPC3) from germinating COS endosperm (Uhrig et al., 2008). Lane 2, 45 μg (A) or 15 μg (B) of protein from the clarified extract of sorghum seeds. Lane 3, 45 μg (A) or 15 μg (B) of 25–60% (NH₄)₂SO₄ fractions. Lane 4, 8 μg (A) or 4 μg (B) of the butyl-Sepharose fraction. Lane 5, 2 μg (A) or 0.1 μg (B) of the DEAE-Fractogel fraction. Lane 6, 2 μg (A) or 0.1 μg (B) of Superdex 200 fractions. ‘M’ denotes various protein molecular weight standards. (C) N-terminal immunocharacterization of the purified PEPC from 48h germinated sorghum seeds. Samples were subjected to 8% SDS–PAGE, blot-transferred onto a PVDF membrane, and probed with anti-N24, anti-SIDAQLR, anti-C19, and anti-pSer13. All lanes contain 0.6 μg of protein except lane anti-C19 that contains 0.2 μg.

Fig. 4.

Incubation with the deubiquitinating enzyme USP-2 core converts the p110:p107 heterotetrameric PEPC from germinated sorghum seeds into a p107 homotetramer. Purified germinated sorghum seed PEPC was incubated in the presence or absence of 20 μM USP-2 core, at 30 ºC. Aliquots were removed at various times and subjected to immunoblot analysis using (A and C) anti-UB (250ng PEPC lane^–1) or (B) anti-COS PTPC (25ng PEPC lane^–1).

Fig. 5.

The p110 subunit of purified PEPC from germinated sorghum seeds is monoubiquitinated at Lys624. (A) TripleTOF MS/MS collision-induced dissociation (CID) analysis of the quadruply charged peptide ion of m/z 469.2522. (B) Orbitrap MS/MS high-energy collision-induced dissociation (HCD) analysis of the triply charged peptide ion of m/z 525.94. C- and N-terminal fragment ions are denoted by y and b, respectively.

Fig. 6.

PEPC monoubiquitination in developing sorghum seeds. (A), Developmental stages of seed considered in this work, I (4–12), II (13–15), III (16–20), IV (21–27), V (28–30), and VI (31–40) days post-anthesis. (B) Clarified extracts from developing seeds were incubated in the presence and absence of 5 μM USP-2 core for 30min and subjected to immunoblot analysis using anti-COS PTPC (45 μg protein lane^–1).