Bacterial‐ and plant‐type phosphoenolpyruvate carboxylase polypeptides interact in the hetero‐oligomeric Class‐2 PEPC complex of developing castor oil seeds

Abstract

SummaryTwo classes of phosphoenolpyruvate carboxylase (PEPC) sharing the same 107-kDa catalytic subunit (p107) were previously purified from developing castor oil seed (COS) endosperm. The association of p107 with an immunologically unrelated 64-kDa polypeptide (p64) causes pronounced physical and kinetic differences between the Class-1 PEPC p107 homotetramer and Class-2 PEPC p107/p64 hetero-octamer. Tryptic peptide sequencing matched p64 to the deduced C-terminal half of several bacterial-type PEPCs (BTPCs) of vascular plants. Immunoblots probed with anti-(COS p64 peptide or p107)-IgG established that: (i) BTPC exists in vivo as an approximately 118-kDa polypeptide (p118) that is rapidly truncated to p64 by an endogenous cysteine endopeptidase during incubation of COS extracts on ice, and (ii) mature and germinated COS contain Class-1 PEPC and p107, but no detectable Class-2 PEPC nor p118. Non-denaturing PAGE, in-gel PEPC activity staining and immunoblotting of developing COS extracts demonstrated that p118 and p107 are subunits of the nonproteolysed approximately 910-kDa Class-2 PEPC complex. As total PEPC activity of clarified COS extracts was unaffected following p118 truncation to p64, the BTPC p118 may function as a regulatory rather than catalytic subunit of the Class-2 PEPC. Moreover, recombinant AtPPC3 and AtPPC4 (Arabidopsis orthologs of COS p107 and p118) expressed as active and inactive PEPCs, respectively. Cloning of cDNAs encoding p118 (RcPpc4) and p107 (RcPpc3) confirmed their respective designation as bacterial-and plant-type PEPCs. Levels of RcPpc3 and RcPpc4 transcripts generally mirrored the respective amounts of p107 and p118. The collective findings provide insights into the molecular features and functional significance of vascular plant BTPCs.

Reciprocal Control of Anaplerotic Phosphoenolpyruvate Carboxylase by in Vivo Monoubiquitination and Phosphorylation in Developing Proteoid Roots of Phosphate-Deficient Harsh Hakea

“…Peptide mass fingerprinting also confirmed that the copurifying p93 is SUS (Supplemental Table S3). Extensive copurification of developing COS Class-1 PEPC with SUS also occurs (Gennidakis et al, 2007). Future research is needed to assess the intriguing possibility that these two key enzymes of cytosolic carbohydrate metabolism might physically interact in vivo.…”

“…The deduced sequence contains: (1) the C-terminal QNTG tetrapeptide characteristic of PTPCs, (2) exact matches to MS/MS derived sequences determined for all four tryptic peptides obtained from the p110 and p107 subunits of the PEPC purified from stage I and II proteoid roots (Fig. 5C), and (3) Ser and Lys residues that align with the positions of the phosphorylatedSer-11 and monoubiquitinated-Lys-628 of developing and germinating COS PTPC (RcPPC3), respectively, and that appear to be absolutely conserved in all PTPCs (Tripodi et al, 2005;Gennidakis et al, 2007;Uhrig et al, 2008;O'Leary et al, 2011a). Research is in progress to isolate a full-length PTPC complementary DNA from proteoid roots of -Pi harsh hakea to determine if its deduced amino acid sequence matches that derived by sequencing the transcriptome of nonproteoid roots.…”

“…All gel and immunoblot results were replicated a minimum of three times with representative results shown in the various figures. Preparation of rabbit anti-COS PEPC and pSer11 has been described elsewhere (Tripodi et al, 2005;Gennidakis et al, 2007). For anti-pSer11 immunoblots the corresponding dephosphopeptide (10 mg mL 21 ) was used to block any nonspecific cross reaction with nonphosphorylated PEPC (Tripodi et al, 2005).…”

The Dual-Targeted Purple Acid Phosphatase Isozyme AtPAP26 Is Essential for Efficient Acclimation of Arabidopsis to Nutritional Phosphate Deprivation

“…All immunoblot results were replicated a minimum of three times, with representative results shown in the various figures. Nondenaturing PAGE was carried out using 7% separating gels (Gennidakis et al, 2007). In-gel APase activity staining was performed by equilibrating the gels in 100 mM Na acetate (pH 5.6) containing 10 mM MgCl 2 for 30 min, and then incubating in equilibration buffer containing 1 mg mL 21 Fast Garnet GBC and 0.03% (w/v) b-naphthyl-P.…”

“…Normalization of RNA for RT was performed for each sample by density measurement of 28S ribosomal RNA bands from the above gel (scanned using ImageJ software from the National Institutes for Health). RNA (5 mg) was reverse transcribed with Superscript III (Invitrogen) and noncompetitive RT-PCR was performed as described in Gennidakis et al (2007). Primers used to amplify AtPPCK1, AtPAP17, AtPAP26, and AtACT2 were previously described (Veljanovski et al, 2006;Gregory et al, 2009).…”