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Published on January 9, 2008; 10.1104/pp.107.110361

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Received October 3, 2007
Accepted December 30, 2007

Co-immunopurification of phosphorylated bacterial- and plant-type phosphoenolpyruvate carboxylases with the plastidial pyruvate dehydrogenase complex from developing castor oil seeds

R. Glen Uhrig , Brendan O'Leary , H. Elizabeth Spang , Justin A. MacDonald , Yi-Min She , and William C. Plaxton *

Department of Biology, Department of Chemistry, and Department of Biochemistry, Queen's University, Kingston, Ontario, Canada K7L 3N6; and Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada T2N 1N4

* Corresponding author; email: plaxton{at}queensu.ca.

The phosphoenolpyruvate carboxylase (PEPC) interactome of developing castor oil seed (COS) (Ricinus communis) endosperm was assessed using co-immunopurification (co-IP) followed by proteomic analysis. Earlier studies suggested that immunologically unrelated 107-kDa plant-type and 118-kDa bacterial-type PEPCs (p107/PTPC and p118/BTPC, respectively) are subunits of an unusual 910-kDa hetero-octameric Class-2 PEPC complex of developing COS. The current results demonstrate that a tight physical interaction occurs between p118 and p107, since p118 quantitatively co-IP'd with p107 following elution of COS extracts through an anti-p107-IgG immunoaffinity column. PEPC activity or immunoreactive PEPC polypeptides were undetectable in the corresponding flow-through fractions. Although BTPCs lack the N-terminal phosphorylation site characteristic of PTPCs, Pro-Q Diamond phosphoprotein staining, immunoblotting with phospho-(Ser/Thr) Akt substrate IgG, and phosphate-affinity PAGE established that co-IP'd p118 was multi-phosphorylated at unique Ser and/or Thr residue(s). Tandem mass spectrometric analysis of an endoproteinase Lys-C p118 peptide digest demonstrated that Ser425 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. As 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-kDa PTPC co-IP'd with p118 and p107 when depodded COS was used. The plastidial pyruvate dehydrogenase complex (PDCpl) was identified as a novel PEPC interactor. Thus, a putative metabolon involving PEPC and PDCpl could function to channel carbon from phosphoenolpyruvate to acetyl-CoA and/or to recycle CO2 from PDCpl to PEPC.




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