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.

Bacterial-type Phosphoenolpyruvate Carboxylase (PEPC) Functions as a Catalytic and Regulatory Subunit of the Novel Class-2 PEPC Complex of Vascular Plants

“…Tryptic peptide sequencing revealed that the 64-kDa polypeptides matched the C-terminal half of deduced rice and Arabidopsis ϳ116-kDa BTPC polypeptides (10). Subsequent studies confirmed that the 64-kDa subunit was derived from a larger 118-kDa polypeptide via in vitro proteolysis, and that native COS Class-2 PEPC exists as a 910-kDa complex consisting of the Class-1 PEPC (RcPPC3) homotetrameric core tightly associated with four 118-kDa BTPC subunits (encoded by RcPpc4; GenBank accession number EF634318) that are in vivo phosphorylated at multiple sites (11,14). Nevertheless, the interaction of RcPPC3 with truncated RcPPC4 polypeptides in the purified native Class-2 PEPC resulted in significant physical and kinetic differences between the COS Class-1 versus Class-2 PEPC that were remarkably analogous to the respective properties of Class-1 and Class-2 PEPCs from unicellular green algae.…”

“…Nevertheless, the interaction of RcPPC3 with truncated RcPPC4 polypeptides in the purified native Class-2 PEPC resulted in significant physical and kinetic differences between the COS Class-1 versus Class-2 PEPC that were remarkably analogous to the respective properties of Class-1 and Class-2 PEPCs from unicellular green algae. In particular, COS and green algal high M r Class-2 PEPC complexes are largely desensitized to metabolite effectors and arise from a tight interaction between unrelated PTPC and BTPC polypeptides (10,11,(13)(14)(15)(16)(17)(18)19). The combined data imply that Class-1 and Class-2 PEPC oligomers evolved in green algae prior to the evolution of vascular plants, with this feature being conserved as a key structure-function aspect of certain vascular plant PEPCs.…”

“…Recombinant RcPPC4 Forms Class-2 PEPCs When Combined with Class-1 PEPCs-Although COS and green algal BTPCs are catalytically active several independent lines of evidence strongly suggest that the native BTPCs only exist in vivo in physical association with corresponding PTPC subunits in a Class-2 PEPC enzyme complex (10,11,(13)(14)(15)(16)(17)(18)19). It is notable that throughout its purification the recombinant RcPPC4 tends to self-associate into large aggregates of limited solubility, which precluded the use of biophysical techniques for assessing its interaction with Class-1 PEPCs.…”

“…Co-immunopurification-This was performed as previously described (14). A clarified extract from 5 g of stage V (midcotyledon) developing COS endosperm was eluted at 0.5 ml/min through a column containing 1.5 mg of protein A-purified anti-RcPPC4-IgG that had been covalently coupled to 1 ml of AminoLink plus gel (Pierce Chemicals) as per the manufacturer's instructions.…”

“…A functional BTPC enzyme has yet to be identified in vascular plants, although BTPC transcripts and genes have been well documented (7-9, 11, 12). Most insights into the occurrence, structure, and function of plant BTPC polypeptides have arisen from the biochemical characterization of unusual Class-2 PEPC hetero-oligomeric complexes from developing COS and unicellular green algae (10,11,(13)(14)(15)(16)(17)(18)19), as well as an active recombinant BTPC from the green alga Chlamydomonas reinhardtii (20). During PEPC purification from the triglyceride-rich endosperm of developing COS, low and high M r isoforms corresponding to a Class-1 PEPC and a novel hetero-octameric Class-2 PEPC were isolated and characterized (10).…”