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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Coimmunopurification of Phosphorylated Bacterial- and Plant-Type Phosphoenolpyruvate Carboxylases with the Plastidial Pyruvate Dehydrogenase Complex from Developing Castor Oil Seeds^1,2,[W],[OA]

Department of Biology (R.G.U., B.O., H.E.S., W.C.P.), Department of Chemistry (Y.-M.S.), and Department of Biochemistry (W.C.P.), Queen's University, Kingston, Ontario, Canada K7L 3N6; and Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada T2N 4N1 (J.A.M.)

The phosphoenolpyruvate carboxylase (PEPC) interactome of developing castor oil seed (COS; Ricinus communis) endosperm was assessed using coimmunopurification (co-IP) followed by proteomic analysis. Earlier studies suggested that immunologically unrelated 107-kD plant-type PEPCs (p107/PTPC) and 118-kD bacterial-type PEPCs (p118/BTPC) are subunits of an unusual 910-kD hetero-octameric class 2 PEPC complex of developing COS. The current results confirm that a tight physical interaction occurs between p118 and p107 because p118 quantitatively coimmunopurified with p107 following elution of COS extracts through an anti-p107-IgG immunoaffinity column. No PEPC activity or immunoreactive PEPC polypeptides were detected in the corresponding flow-through fractions. Although BTPCs lack the N-terminal phosphorylation motif characteristic of PTPCs, Pro-Q Diamond phosphoprotein staining, immunoblotting with phospho-serine (Ser)/threonine Akt substrate IgG, and phosphate-affinity PAGE established that coimmunopurified p118 was multiphosphorylated at unique Ser and/or threonine residues. Tandem mass spectrometric analysis of an endoproteinase Lys-C p118 peptide digest demonstrated that Ser-425 is subject to in vivo proline-directed phosphorylation. The co-IP of p118 with p107 did not appear to be influenced by their phosphorylation status. Because p118 phosphorylation was unchanged 48 h following elimination of photosynthate supply due to COS depodding, the signaling mechanisms responsible for photosynthate-dependent p107 phosphorylation differ from those controlling p118's in vivo phosphorylation. A 110-kD PTPC coimmunopurified with p118 and p107 when depodded COS was used. The plastidial pyruvate dehydrogenase complex (PDC_pl) was identified as a novel PEPC interactor. Thus, a putative metabolon involving PEPC and PDC_pl could function to channel carbon from phosphoenolpyruvate to acetyl-coenzyme A and/or to recycle CO₂ from PDC_pl to PEPC.

² This article is dedicated to the distinguished plant biochemist and PEPC researcher Prof. Raymond Chollet (University of Nebraska) on the occasion of his retirement.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: William C. Plaxton (plaxton{at}queensu.ca).

^[W] The online version of this article contains Web-only data.

^[OA] Open Access articles can be viewed online without a subscription.

www.plantphysiol.org/cgi/doi/10.1104/pp.107.110361

^* Corresponding author; e-mail plaxton{at}queensu.ca.

Received October 3, 2007; accepted December 30, 2007; published January 9, 2008.