Abstract
Phosphoenolpyruvate carboxylase (PEPC) is believed to play an important role in producing malate as a substrate for fatty acid synthesis by leucoplasts of the developing castor oilseed (COS) endosperm. Two kinetically distinct isoforms of COS PEPC were resolved by gel filtration chromatography and purified. PEPC1 is a typical 410-kDa homotetramer composed of 107-kDa subunits (p107). In contrast, PEPC2 exists as an unusual 681-kDa hetero-octamer composed of the same p107 found in PEPC1 and an associated 64-kDa polypeptide (p64) that is structurally and immunologically unrelated to p107. Relative to PEPC1, PEPC2 demonstrated significantly enhanced thermal stability and a much lower sensitivity to allosteric activators (Glc-6-P, Glc-1-P, Fru-6-P, glycerol-3-P) and inhibitors (Asp, Glu, malate) and pH changes within the physiological range. Nondenaturing PAGE of clarified extracts followed by in-gel PEPC activity staining indicated that the ratio of PEPC1:PEPC2 increases during COS development such that only PEPC1 is detected in mature COS. Dissimilar developmental profiles and kinetic properties support the hypotheses that (i) PEPC1 functions to replenish dicarboxylic acids consumed through transamination reactions required for storage protein synthesis, whereas (ii) PEPC2 facilitates PEP flux to malate in support of fatty acid synthesis. Interestingly, the respective physical and kinetic properties of COS PEPC1 and PEPC2 are remarkably comparable with those of the homotetrameric low M(r) Class 1 and heteromeric high M(r) Class 2 PEPC isoforms of unicellular green algae.
Highlights
Pates in photosynthesis by catalyzing the initial fixation of atmospheric CO2
Dissimilar developmental profiles and kinetic properties support the hypotheses that (i) PEPC1 functions to replenish dicarboxylic acids consumed through transamination reactions required for storage protein synthesis, whereas (ii) PEPC2 facilitates PEP flux to malate in support of fatty acid synthesis
We provide evidence that the association of a common 107-kDa PEPC catalytic subunit with an unrelated but PEPC-like 64-kDa polypeptide is responsible for the dramatic differences in the physical
Summary
Plant Material—Castor plants (Ricinus communis L., cv Baker 296) were cultivated in a greenhouse at 24 °C and 60% relative humidity under natural light supplemented with 16 h of artificial light. PEG pellets were dissolved in buffer D to a protein concentration of approximately 10 mg/ml, centrifuged as described above, and loaded at 0.6 ml/min onto a column (1.1 ϫ 12 cm) of Fractogel EMD DEAE-650 (S) that had been connected to the FPLC and equilibrated with buffer D. Each PEPC pool was concentrated to approximately 0.2 ml and applied separately at 0.2 ml/min onto a Superose-6 HR 10/30 column equilibrated with buffer E. Samples destined for ESI/Q-TOF MS/MS were applied automatically by capillary liquid chromatography to a Micromass Q-TOF Ultima GLOBAL using a C18 column (75 m ϫ 150 mm) running at 200 nl/min Mass data from both machines were used to search the NCBInr data base using MASCOT (www.matrixscience.com)
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