In vitro phosphorylation of phosphoenolpyruvate carboxylase from the green alga Selenastrum minutum

• Published in: Plant and cell physiology 2002-07, Vol.43 (7), p.785-792
• Main Authors: Rivoal, J. (University of Manitoba, Winnipeg (Canada)), Turpin, D.H, Plaxton, W.C
• Format: Article
• Language: English
• Subjects: CHLOROPHYCEAE; Chlorophyta - enzymology; CHROMATOGRAPHY; fast protein liquid chromatography; FPLC; IN VITRO EXPERIMENTATION; Isoenzymes - isolation & purification; Isoenzymes - metabolism; Keywords: Green algae — isoform — Phosphoenolpyruvate carboxylase — Protein phosphorylation — Protein structure — Selenastrum minutum; LYASES; Microcystins; PEP; PEPC; Peptides, Cyclic - pharmacology; phosphoenolpyruvate; phosphoenolpyruvate carboxylase (EC 4.1.1.31); Phosphoenolpyruvate Carboxylase - chemistry; Phosphoenolpyruvate Carboxylase - isolation & purification; Phosphoenolpyruvate Carboxylase - metabolism; Phosphoric Monoester Hydrolases - antagonists & inhibitors; PHOSPHORYLATION; Phosphorylation - drug effects; PP2A; PROTEIN KINASE; Protein Kinases - metabolism; protein phosphatase 2A; PROTEINS; PYRUVATES; Selenastrum minutum; Sodium Fluoride - pharmacology
• ISSN: 0032-0781; 1471-9053
• DOI: 10.1093/pcp/pcf092

Previously, we described two distinct classes of phosphoenolpyruvate carboxylase (PEPC) isoforms in the green alga Selenastrum minutum. Class 1 PEPC (PEPC1) is a homotetramer composed of 102 kDa subunits (p102), whereas Class 2 PEPCs exist as three large protein complexes (PEPC2-PEPC4) containing varying proportions of structurally dissimilar p102 and 130 kDa (p130) PEPC catalytic subunits. In the current study, a p102 calciwnindependent protein kinase was shown to co-purify with PEPC1, but not PEPC2. However, the p130 subunit of PEPC2 was phosphorylated in vitro during its incubation in the presence of [gamma- sup32 P]ATP and a clarified algal extract. Treatment of purified PEPC2 with protein phosphatase 2A sub2 increased its apparent M sub(r) as judged by Superose 6 gel filtration chromatography. The presence of the protein phosphatase inhibitors NaF and microcystin-LR throughout PEPC purification significantly influenced the activity and structural organization of Class 2, but not Class 1, PEPC isoforms. The results are consistent with the notion that under the culture conditions employed: (i) Class 1 and Class 2 PEPC isoforms exist in vivo mainly in their dephosphorylated and phosphorylated forms, respectively, and (ii) phosphorylation of Class 2 PEPCs Ieads to a significant reduction in their activity and native M sub(r) . We propose that protein kinase-mediated phosphorylation is involved in the control and structural organization of green algal PEPC.