Journal Article

In Vitro Phosphorylation of Phospho<i>enol</i>pyruvate Carboxylase from the Green Alga <i>Selenastrum minutum</i>

Jean Rivoal, David H. Turpin and William C. Plaxton

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 43, issue 7, pages 785-792
Published in print July 2002 | ISSN: 0032-0781
Published online July 2002 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pcf092
In Vitro Phosphorylation of Phosphoenolpyruvate Carboxylase from the Green Alga Selenastrum minutum

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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 calcium-independent 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 [γ-32P]ATP and a clarified algal extract. Treatment of purified PEPC2 with protein phosphatase 2A2 increased its apparent Mr 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 leads to a significant reduction in their activity and native Mr. We propose that protein kinase-mediated phosphorylation is involved in the control and structural organization of green algal PEPC.

Keywords: Keywords: Green algae — isoform — Phosphoenolpyruvate carboxylase — Protein phosphorylation — Protein structure — Selenastrum minutum.; Abbreviations: FPLC, fast protein liquid chromatography; PEP, phosphoenolpyruvate; PEPC, phosphoenolpyruvate carboxylase (EC 4.1.1.31); PP2A, protein phosphatase 2A.

Journal Article.  5333 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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