title: Coimmunopurification of phosphorylated bacterial- and plant-type phosphoenolpyruvate carboxylases with the plastidial pyruvate dehydrogenase complex from developing castor oil seeds.
abstract: 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 CO(2) from PDC(pl) to PEPC.
authors.affiliation: Department of Biology, Queen's University, Kingston, ON, Canada.
authors.forename: R Glen
authors.initials: RG
authors.lastname: Uhrig
authors.forename: Brendan
authors.initials: B
authors.lastname: O'Leary
authors.forename: H Elizabeth
authors.initials: HE
authors.lastname: Spang
authors.forename: Justin A
authors.initials: JA
authors.lastname: MacDonald
authors.forename: Yi-Min
authors.initials: YM
authors.lastname: She
authors.forename: William C
authors.initials: WC
authors.lastname: Plaxton
mesh_terms: D000595:Amino Acid Sequence; D002367:Ricinus communis / Q000201:enzymology* / Q000254:growth & development; D047468:Immunoprecipitation; D007527:Isoenzymes / Q000378:metabolism; D008969:Molecular Sequence Data; D010730:Phosphoenolpyruvate Carboxylase / Q000302:isolation & purification / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D018087:Plastids / Q000201:enzymology*; D025941:Protein Interaction Mapping; D040901:Proteomics; D011768:Pyruvate Dehydrogenase Complex / Q000302:isolation & purification / Q000378:metabolism*; D012639:Seeds / Q000201:enzymology* / Q000254:growth & development; D012694:Serine / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D010750:Phosphoproteins; D011768:Pyruvate Dehydrogenase Complex; D012694:Serine; D010730:Phosphoenolpyruvate Carboxylase