Title: Structural and Kinetic Properties of High and Low Molecular Mass Phosphoenolpyruvate Carboxylase Isoforms from the Endosperm of Developing Castor Oilseeds
Abstract: Phosphoenolpyruvate carboxylase (PEPC) is believed to play an important role in producing malate as a substrate for fatty acid synthesis by leucoplasts of the developing castor oilseed (COS) endosperm. Two kinetically distinct isoforms of COS PEPC were resolved by gel filtration chromatography and purified. PEPC1 is a typical 410-kDa homotetramer composed of 107-kDa subunits (p107). In contrast, PEPC2 exists as an unusual 681-kDa hetero-octamer composed of the same p107 found in PEPC1 and an associated 64-kDa polypeptide (p64) that is structurally and immunologically unrelated to p107. Relative to PEPC1, PEPC2 demonstrated significantly enhanced thermal stability and a much lower sensitivity to allosteric activators (Glc-6-P, Glc-1-P, Fru-6-P, glycerol-3-P) and inhibitors (Asp, Glu, malate) and pH changes within the physiological range. Nondenaturing PAGE of clarified extracts followed by in-gel PEPC activity staining indicated that the ratio of PEPC1:PEPC2 increases during COS development such that only PEPC1 is detected in mature COS. Dissimilar developmental profiles and kinetic properties support the hypotheses that (i) PEPC1 functions to replenish dicarboxylic acids consumed through transamination reactions required for storage protein synthesis, whereas (ii) PEPC2 facilitates PEP flux to malate in support of fatty acid synthesis. Interestingly, the respective physical and kinetic properties of COS PEPC1 and PEPC2 are remarkably comparable with those of the homotetrameric lowMr Class 1 and heteromeric highMr Class 2 PEPC isoforms of unicellular green algae. Phosphoenolpyruvate carboxylase (PEPC) is believed to play an important role in producing malate as a substrate for fatty acid synthesis by leucoplasts of the developing castor oilseed (COS) endosperm. Two kinetically distinct isoforms of COS PEPC were resolved by gel filtration chromatography and purified. PEPC1 is a typical 410-kDa homotetramer composed of 107-kDa subunits (p107). In contrast, PEPC2 exists as an unusual 681-kDa hetero-octamer composed of the same p107 found in PEPC1 and an associated 64-kDa polypeptide (p64) that is structurally and immunologically unrelated to p107. Relative to PEPC1, PEPC2 demonstrated significantly enhanced thermal stability and a much lower sensitivity to allosteric activators (Glc-6-P, Glc-1-P, Fru-6-P, glycerol-3-P) and inhibitors (Asp, Glu, malate) and pH changes within the physiological range. Nondenaturing PAGE of clarified extracts followed by in-gel PEPC activity staining indicated that the ratio of PEPC1:PEPC2 increases during COS development such that only PEPC1 is detected in mature COS. Dissimilar developmental profiles and kinetic properties support the hypotheses that (i) PEPC1 functions to replenish dicarboxylic acids consumed through transamination reactions required for storage protein synthesis, whereas (ii) PEPC2 facilitates PEP flux to malate in support of fatty acid synthesis. Interestingly, the respective physical and kinetic properties of COS PEPC1 and PEPC2 are remarkably comparable with those of the homotetrameric lowMr Class 1 and heteromeric highMr Class 2 PEPC isoforms of unicellular green algae. PEP carboxylase (EC 4.1.1.31) phosphoenolpyruvate crassulacean acid metabolism castor oilseed electrospray quadrupole-time of flight tandem mass spectrometry matrix-assisted laser desorption ionization-time of flight mass spectrometry fast protein liquid chromatography polyethylene glycol 4-morpholineethanesulfonic acid Phosphoenolpyruvate carboxylase (PEPC)1 is a ubiquitous cytosolic enzyme in vascular plants that is also widely distributed in green algae and bacteria (1Chollet R. Vidal J. O' Leary M.H. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 273-298Google Scholar). It catalyzes the irreversible ॆ-carboxylation of PEP in the presence of Mg2+ and HCO3− to yield oxaloacetate and Pi. PEPC is abundant in C4 and crassulacean acid metabolism (CAM) leaves where it participates in photosynthesis by catalyzing the initial fixation of atmospheric CO2. Both allosteric mechanisms and covalent modification are involved in PEPC control in C4 and CAM leaves (1Chollet R. Vidal J. O' Leary M.H. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 273-298Google Scholar, 2Rajagopalan A., V Devi M.T. Raghavendra A.S. Photosynth. Res. 1994; 39: 115-135Google Scholar, 3Nimmo H.G. Batey N.H. Dickinson H.G. Hetherington S.M. Society for Experimental Biology Seminar Series 53: Post Translational Modifications in Plants. Cambridge University Press, Cambridge, United Kingdom1993: 161-170Google Scholar). Early work established that C4 and CAM PEPCs are controlled by a diurnal cycle that modulates their sensitivity to l-malate inhibition (1Chollet R. Vidal J. O' Leary M.H. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 273-298Google Scholar, 2Rajagopalan A., V Devi M.T. Raghavendra A.S. Photosynth. Res. 1994; 39: 115-135Google Scholar, 3Nimmo H.G. Batey N.H. Dickinson H.G. Hetherington S.M. Society for Experimental Biology Seminar Series 53: Post Translational Modifications in Plants. Cambridge University Press, Cambridge, United Kingdom1993: 161-170Google Scholar). This cycle is the result of phosphorylation of the PEPC homotetramer by an endogenous Ca2+-independent PEPC protein kinase and dephosphorylation by a protein phosphatase type 2A at a highly conserved seryl residue localized near the N terminus of the 100–110-kDa PEPC subunit (1Chollet R. Vidal J. O' Leary M.H. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 273-298Google Scholar, 2Rajagopalan A., V Devi M.T. Raghavendra A.S. Photosynth. Res. 1994; 39: 115-135Google Scholar, 3Nimmo H.G. Batey N.H. Dickinson H.G. Hetherington S.M. Society for Experimental Biology Seminar Series 53: Post Translational Modifications in Plants. Cambridge University Press, Cambridge, United Kingdom1993: 161-170Google Scholar).Relative to C4 and CAM PEPCs, the properties of the enzyme from non-green plant tissues are less well understood. Although proposed roles for nonphotosynthetic PEPCs are diverse, a crucial PEPC function is the anaplerotic replenishment of citric acid cycle intermediates consumed during biosynthesis and nitrogen assimilation (1Chollet R. Vidal J. O' Leary M.H. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 273-298Google Scholar). As with C4 and CAM PEPCs, the PEPC of C3leaves and nonphotosynthetic tissues can be controlled by allosteric effectors and reversible phosphorylation (4Duff S.M.G. Chollet R. Plant Physiol. 1995; 107: 775-782Google Scholar, 5Munoz T. Escribano M.I. Merodio C. Phytochemistry. 2001; 58: 1007-1013Google Scholar, 6Schuller K.A. Turpin D.H. Plaxton W.C. Plant Physiol. 1990; 94: 1429-1435Google Scholar, 7Zhang X.Q. Chollet R. Arch. Biochem. Biophys. 1997; 343: 260-268Google Scholar, 8Moraes T.F. Plaxton W.C. Eur. J. Biochem. 2000; 267: 4465-4476Google Scholar, 9Law R.D. Plaxton W.C. Biochem. J. 1995; 307: 807-816Google Scholar, 10Law R.D. Plaxton W.C. Eur. J. Biochem. 1997; 247: 642-651Google Scholar). However, despite the probable central role of PEPCs in the metabolism of developing and germinating seeds (11Podestá F.E. Plaxton W.C. Planta. 1992; 194: 381-387Google Scholar, 12Hedley C.L. Harvey D.M. Keely R.J. Nature. 1975; 258: 352-354Google Scholar, 13Macnicol P.K. Raymond P. Physiol. Plant. 1998; 103: 132-138Google Scholar, 14Gonzalez M.C. Osuna L. Echevarria C. Vidal J. Cejudo F.J. Plant Physiol. 1998; 116: 1249-1258Google Scholar, 15Golombek S. Heim U. Horstmann C. Wobus U. Weber H. Planta. 1999; 208: 66-72Google Scholar, 16Sangwan R.S. Singh N. Plaxton W.C. Plant Physiol. 1992; 99: 445-449Google Scholar), no seed PEPC has been fully purified and thoroughly characterized.Storage lipids account for as much as 657 of the weight of mature castor oilseeds (COS). Triacylglyceride accumulation depends on the synthesis of long chain fatty acids, which in developing oilseeds occurs in specialized plastids termed leucoplasts. This process requires the transport of both sucrose-derived carbon skeletons and energetic intermediates across the plastid envelope (17Rawsthorne S. Prog. Lipid Res. 2001; 41: 182-196Google Scholar).l-Malate supports significant rates of fatty acid synthesis by isolated leucoplasts from developing COS (18Smith R.G. Gauthier D.A. Dennis D.T. Turpin D.H. Plant Physiol. 1992; 98: 1233-1238Google Scholar). Malate imported from the cytosol into the leucoplast stroma is mediated by a malate/Pi translocator within the COS leucoplast envelope (19Eastmond P.J. Dennis D.T. Rawsthorne S. Plant Physiol. 1997; 114: 851-856Google Scholar). Sangwan and co-workers (16Sangwan R.S. Singh N. Plaxton W.C. Plant Physiol. 1992; 99: 445-449Google Scholar) hypothesized that the large increase in PEPC activity and concentration that accompanies COS development facilitates malate production for fatty acid synthesis. The increased PEP to malate flux would also serve as an anaplerotic source of C-skeletons for transamination reactions associated with COS storage protein synthesis.The aim of this study was to purify and characterize PEPC from developing COS. Here we present unexpected evidence for two PEPC isoforms from developing COS and examine their structural and kinetic properties. Although one isoform is a typical PEPC homotetramer, the other represents a unique high Mr PEPC complex unprecedented in vascular plants but remarkably reminiscent of Class 2 PEPC isoforms recently described in unicellular green algae (20Rivoal J. Dunford R. Plaxton W.C. Turpin D.H. Arch. Biochem. Biophys. 1996; 332: 47-57Google Scholar, 21Rivoal J. Plaxton W.C. Turpin D.H. Biochem. J. 1998; 331: 201-209Google Scholar, 22Rivoal J. Trzos S. Gage D.A. Plaxton W.C. Turpin D.H. J. Biol. Chem. 2001; 276: 12588-12597Google Scholar, 23Rivoal J. Turpin D.H. Plaxton W.C. Plant Cell Physiol. 2002; 43: 785-792Google Scholar). We provide evidence that the association of a common 107-kDa PEPC catalytic subunit with an unrelated but PEPC-like 64-kDa polypeptide is responsible for the dramatic differences in the physical and kinetic properties observed between the PEPC homotetramer and highMr PEPC complex of developing COS.DISCUSSIONWhen partial in vitro proteolysis of p107 was prevented, two COS PEPC isoforms that significantly differed in their physical and kinetic properties were resolved by Superdex 200 FPLC and highly purified. Tissue- and/or developmentally specific PEPC isozymes are known to occur in vascular plants (1Chollet R. Vidal J. O' Leary M.H. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 273-298Google Scholar, 2Rajagopalan A., V Devi M.T. Raghavendra A.S. Photosynth. Res. 1994; 39: 115-135Google Scholar, 3Nimmo H.G. Batey N.H. Dickinson H.G. Hetherington S.M. Society for Experimental Biology Seminar Series 53: Post Translational Modifications in Plants. Cambridge University Press, Cambridge, United Kingdom1993: 161-170Google Scholar), and genetic evidence indicates that developing Glycine max (soybean) andVicia faba seeds express more than one PEPC gene (15Golombek S. Heim U. Horstmann C. Wobus U. Weber H. Planta. 1999; 208: 66-72Google Scholar, 32Sugimoto T. Kawasaki T. Kato T. Whittier R.F. Shibata D. Kawamura Y. Plant Mol. Biol. 1993; 20: 743-747Google Scholar,33Vazquez-Tello A. Whittier R.F. Kawasaki T. Sugimoto T. Kawamura Y. Shibata D. Plant Physiol. 1993; 103: 1025-1026Google Scholar). To our knowledge, this is the first report of the isolation of two PEPC isoforms from the same plant tissue.COS PEPC1 is a p107 homotetramer, typical of most other plant PEPCs studied to date. By contrast, PEPC2 appears to exist as an unusual 681- kDa hetero-octamer composed of the same p107 found in PEPC1 and an associated p64 that is structurally and immunologically unrelated to p107. Nevertheless, Q-TOF MS/MS analysis of tryptic peptides revealed that p64 is highly similar to two putative PEPCs identified by annotation of the rice and Arabidopsis genomes (Fig. 5). Although they contain conserved regions required for PEPC activity, these putative PEPCs exhibit a unique N-terminal region that lacks the regulatory seryl phosphorylation site thought to be conserved among all plant PEPCs (1Chollet R. Vidal J. O' Leary M.H. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 273-298Google Scholar) and have predicted molecular masses of 102 (rice) and 110 kDa (Arabidopsis). Although it is feasible that p64 represents an in vivo or in vitro degradation product of a larger polypeptide, the data indicate that p64 is a PEPC-like polypeptide that interacts with p107 to give rise to the COS PEPC2 heteromeric complex. This association may either physically block the allosteric sites of p107 or promote an allosteric transition in p107 such that effectors have limited access to their respective sites.COS PEPC1 and PEPC2 Emulate Green Algal 舠Class 1舡 and 舠Class 2舡 PEPC IsoformsInterestingly, the respective properties of COS PEPC1 and PEPC2 are remarkably comparable with those of the homotetrameric low Mr Class 1 and heteromeric high Mr Class 2 PEPC isoforms of the green algaeSelenastrum minutum and Chlamydomonas reinhardtii(20Rivoal J. Dunford R. Plaxton W.C. Turpin D.H. Arch. Biochem. Biophys. 1996; 332: 47-57Google Scholar, 21Rivoal J. Plaxton W.C. Turpin D.H. Biochem. J. 1998; 331: 201-209Google Scholar, 22Rivoal J. Trzos S. Gage D.A. Plaxton W.C. Turpin D.H. J. Biol. Chem. 2001; 276: 12588-12597Google Scholar). All algal PEPC isoforms share the same 102-kDa catalytic subunit (p102). Similar to COS PEPC2, the algal Class 2 PEPCs also contain associated polypeptides that are immunologically unrelated to p102. MALDI-TOF MS and microsequencing revealed that like the p64 of COS PEPC2, the 130- kDa subunit (p130) of S. minutum Class 2 PEPC represents a distinct PEPC-like polypeptide that is only distantly related to p102 (22Rivoal J. Trzos S. Gage D.A. Plaxton W.C. Turpin D.H. J. Biol. Chem. 2001; 276: 12588-12597Google Scholar). Moreover, relative to COS PEPC1 and algal Class 1 PEPCs, the COS PEPC2 and algal Class 2 PEPCs demonstrate significantly enhanced thermal stability and a much lower sensitivity to allosteric effectors and pH changes within the physiological range (20Rivoal J. Dunford R. Plaxton W.C. Turpin D.H. Arch. Biochem. Biophys. 1996; 332: 47-57Google Scholar, 21Rivoal J. Plaxton W.C. Turpin D.H. Biochem. J. 1998; 331: 201-209Google Scholar). Taken together, the data imply that high and low MrPEPC isoforms arose in green algae before the evolution of vascular plants, with this feature being conserved as a key structure-function aspect of at least some plant PEPCs.Possible Functions and Interconversion of COS PEPC1 and PEPC2Similar to PEPCs from other non-green plant tissues (6Schuller K.A. Turpin D.H. Plaxton W.C. Plant Physiol. 1990; 94: 1429-1435Google Scholar, 8Moraes T.F. Plaxton W.C. Eur. J. Biochem. 2000; 267: 4465-4476Google Scholar,9Law R.D. Plaxton W.C. Biochem. J. 1995; 307: 807-816Google Scholar, 11Podestá F.E. Plaxton W.C. Planta. 1992; 194: 381-387Google Scholar, 15Golombek S. Heim U. Horstmann C. Wobus U. Weber H. Planta. 1999; 208: 66-72Google Scholar), COS PEPC1 was activated by hexose-mono-Ps and potently inhibited by malate, Asp, and Glu at pH 7.3 (Table II). This result indicates that PEPC1 may fulfill a key anaplerotic role to replenish dicarboxylic acids consumed through transamination reactions required to support storage protein synthesis. The inhibition of PEPC1 by Asp and Glu provides a tight feedback control that could closely balance PEPC1 activity with the production of C-skeletons (i.e. oxaloacetate, 2-oxoglutarate) required for NH4+ assimilation or transamination reactions. The 舠effector-insensitive舡 PEPC2, by contrast, may facilitate PEP flux to malate in support of leucoplast fatty acid synthesis despite the significant malate levels present in developing COS (18Smith R.G. Gauthier D.A. Dennis D.T. Turpin D.H. Plant Physiol. 1992; 98: 1233-1238Google Scholar). Nondenaturing PAGE of clarified COS extracts followed by in-gel PEPC activity staining revealed that PEPC2 increases during COS development, peaking at stage VII, and then rapidly disappears during COS maturation (Fig.4C). This pattern parallels triglyceride accumulation in this tissue, which also peaks at stage VII (34Simcox P.D. Garland W. DeLuca V. Canvin D.T. Dennis D.T. Can. J. Bot. 1979; 57: 1008-1014Google Scholar). The developmental profile for PEPC1 (Fig. 4C), by contrast, parallels that of storage protein accumulation (24Greenwood J. Bewley J. Can. J. Bot. 1982; 60: 1751-1760Google Scholar), with both becoming maximal during the maturation phase of COS development. Further studies using transgenic plants and/or pharmacological inhibitors will help to fully evaluate the metabolic functions of COS PEPC1 and PEPC2.It remains to be determined whether and how COS PEPC1 and PEPC2 interconvert. However, protein-kinase-mediated phosphorylation of p102 appears to be involved in the control and structural organization of green algal (S. minutum) Class 2 PEPCs (23Rivoal J. Turpin D.H. Plaxton W.C. Plant Cell Physiol. 2002; 43: 785-792Google Scholar). COS p107 contains the N-terminal regulatory seryl phosphorylation site characteristic of most plant PEPCs (Fig. 3A). It will be of interest to determine whether COS PEPC1 and PEPC2 are interconverted via a phosphorylation-dephosphorylation mechanism involving p107 and/or p64. Phosphoenolpyruvate carboxylase (PEPC)1 is a ubiquitous cytosolic enzyme in vascular plants that is also widely distributed in green algae and bacteria (1Chollet R. Vidal J. O' Leary M.H. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 273-298Google Scholar). It catalyzes the irreversible ॆ-carboxylation of PEP in the presence of Mg2+ and HCO3− to yield oxaloacetate and Pi. PEPC is abundant in C4 and crassulacean acid metabolism (CAM) leaves where it participates in photosynthesis by catalyzing the initial fixation of atmospheric CO2. Both allosteric mechanisms and covalent modification are involved in PEPC control in C4 and CAM leaves (1Chollet R. Vidal J. O' Leary M.H. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 273-298Google Scholar, 2Rajagopalan A., V Devi M.T. Raghavendra A.S. Photosynth. Res. 1994; 39: 115-135Google Scholar, 3Nimmo H.G. Batey N.H. Dickinson H.G. Hetherington S.M. Society for Experimental Biology Seminar Series 53: Post Translational Modifications in Plants. Cambridge University Press, Cambridge, United Kingdom1993: 161-170Google Scholar). Early work established that C4 and CAM PEPCs are controlled by a diurnal cycle that modulates their sensitivity to l-malate inhibition (1Chollet R. Vidal J. O' Leary M.H. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 273-298Google Scholar, 2Rajagopalan A., V Devi M.T. Raghavendra A.S. Photosynth. Res. 1994; 39: 115-135Google Scholar, 3Nimmo H.G. Batey N.H. Dickinson H.G. Hetherington S.M. Society for Experimental Biology Seminar Series 53: Post Translational Modifications in Plants. Cambridge University Press, Cambridge, United Kingdom1993: 161-170Google Scholar). This cycle is the result of phosphorylation of the PEPC homotetramer by an endogenous Ca2+-independent PEPC protein kinase and dephosphorylation by a protein phosphatase type 2A at a highly conserved seryl residue localized near the N terminus of the 100–110-kDa PEPC subunit (1Chollet R. Vidal J. O' Leary M.H. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 273-298Google Scholar, 2Rajagopalan A., V Devi M.T. Raghavendra A.S. Photosynth. Res. 1994; 39: 115-135Google Scholar, 3Nimmo H.G. Batey N.H. Dickinson H.G. Hetherington S.M. Society for Experimental Biology Seminar Series 53: Post Translational Modifications in Plants. Cambridge University Press, Cambridge, United Kingdom1993: 161-170Google Scholar). Relative to C4 and CAM PEPCs, the properties of the enzyme from non-green plant tissues are less well understood. Although proposed roles for nonphotosynthetic PEPCs are diverse, a crucial PEPC function is the anaplerotic replenishment of citric acid cycle intermediates consumed during biosynthesis and nitrogen assimilation (1Chollet R. Vidal J. O' Leary M.H. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 273-298Google Scholar). As with C4 and CAM PEPCs, the PEPC of C3leaves and nonphotosynthetic tissues can be controlled by allosteric effectors and reversible phosphorylation (4Duff S.M.G. Chollet R. Plant Physiol. 1995; 107: 775-782Google Scholar, 5Munoz T. Escribano M.I. Merodio C. Phytochemistry. 2001; 58: 1007-1013Google Scholar, 6Schuller K.A. Turpin D.H. Plaxton W.C. Plant Physiol. 1990; 94: 1429-1435Google Scholar, 7Zhang X.Q. Chollet R. Arch. Biochem. Biophys. 1997; 343: 260-268Google Scholar, 8Moraes T.F. Plaxton W.C. Eur. J. Biochem. 2000; 267: 4465-4476Google Scholar, 9Law R.D. Plaxton W.C. Biochem. J. 1995; 307: 807-816Google Scholar, 10Law R.D. Plaxton W.C. Eur. J. Biochem. 1997; 247: 642-651Google Scholar). However, despite the probable central role of PEPCs in the metabolism of developing and germinating seeds (11Podestá F.E. Plaxton W.C. Planta. 1992; 194: 381-387Google Scholar, 12Hedley C.L. Harvey D.M. Keely R.J. Nature. 1975; 258: 352-354Google Scholar, 13Macnicol P.K. Raymond P. Physiol. Plant. 1998; 103: 132-138Google Scholar, 14Gonzalez M.C. Osuna L. Echevarria C. Vidal J. Cejudo F.J. Plant Physiol. 1998; 116: 1249-1258Google Scholar, 15Golombek S. Heim U. Horstmann C. Wobus U. Weber H. Planta. 1999; 208: 66-72Google Scholar, 16Sangwan R.S. Singh N. Plaxton W.C. Plant Physiol. 1992; 99: 445-449Google Scholar), no seed PEPC has been fully purified and thoroughly characterized. Storage lipids account for as much as 657 of the weight of mature castor oilseeds (COS). Triacylglyceride accumulation depends on the synthesis of long chain fatty acids, which in developing oilseeds occurs in specialized plastids termed leucoplasts. This process requires the transport of both sucrose-derived carbon skeletons and energetic intermediates across the plastid envelope (17Rawsthorne S. Prog. Lipid Res. 2001; 41: 182-196Google Scholar).l-Malate supports significant rates of fatty acid synthesis by isolated leucoplasts from developing COS (18Smith R.G. Gauthier D.A. Dennis D.T. Turpin D.H. Plant Physiol. 1992; 98: 1233-1238Google Scholar). Malate imported from the cytosol into the leucoplast stroma is mediated by a malate/Pi translocator within the COS leucoplast envelope (19Eastmond P.J. Dennis D.T. Rawsthorne S. Plant Physiol. 1997; 114: 851-856Google Scholar). Sangwan and co-workers (16Sangwan R.S. Singh N. Plaxton W.C. Plant Physiol. 1992; 99: 445-449Google Scholar) hypothesized that the large increase in PEPC activity and concentration that accompanies COS development facilitates malate production for fatty acid synthesis. The increased PEP to malate flux would also serve as an anaplerotic source of C-skeletons for transamination reactions associated with COS storage protein synthesis. The aim of this study was to purify and characterize PEPC from developing COS. Here we present unexpected evidence for two PEPC isoforms from developing COS and examine their structural and kinetic properties. Although one isoform is a typical PEPC homotetramer, the other represents a unique high Mr PEPC complex unprecedented in vascular plants but remarkably reminiscent of Class 2 PEPC isoforms recently described in unicellular green algae (20Rivoal J. Dunford R. Plaxton W.C. Turpin D.H. Arch. Biochem. Biophys. 1996; 332: 47-57Google Scholar, 21Rivoal J. Plaxton W.C. Turpin D.H. Biochem. J. 1998; 331: 201-209Google Scholar, 22Rivoal J. Trzos S. Gage D.A. Plaxton W.C. Turpin D.H. J. Biol. Chem. 2001; 276: 12588-12597Google Scholar, 23Rivoal J. Turpin D.H. Plaxton W.C. Plant Cell Physiol. 2002; 43: 785-792Google Scholar). We provide evidence that the association of a common 107-kDa PEPC catalytic subunit with an unrelated but PEPC-like 64-kDa polypeptide is responsible for the dramatic differences in the physical and kinetic properties observed between the PEPC homotetramer and highMr PEPC complex of developing COS. DISCUSSIONWhen partial in vitro proteolysis of p107 was prevented, two COS PEPC isoforms that significantly differed in their physical and kinetic properties were resolved by Superdex 200 FPLC and highly purified. Tissue- and/or developmentally specific PEPC isozymes are known to occur in vascular plants (1Chollet R. Vidal J. O' Leary M.H. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 273-298Google Scholar, 2Rajagopalan A., V Devi M.T. Raghavendra A.S. Photosynth. Res. 1994; 39: 115-135Google Scholar, 3Nimmo H.G. Batey N.H. Dickinson H.G. Hetherington S.M. Society for Experimental Biology Seminar Series 53: Post Translational Modifications in Plants. Cambridge University Press, Cambridge, United Kingdom1993: 161-170Google Scholar), and genetic evidence indicates that developing Glycine max (soybean) andVicia faba seeds express more than one PEPC gene (15Golombek S. Heim U. Horstmann C. Wobus U. Weber H. Planta. 1999; 208: 66-72Google Scholar, 32Sugimoto T. Kawasaki T. Kato T. Whittier R.F. Shibata D. Kawamura Y. Plant Mol. Biol. 1993; 20: 743-747Google Scholar,33Vazquez-Tello A. Whittier R.F. Kawasaki T. Sugimoto T. Kawamura Y. Shibata D. Plant Physiol. 1993; 103: 1025-1026Google Scholar). To our knowledge, this is the first report of the isolation of two PEPC isoforms from the same plant tissue.COS PEPC1 is a p107 homotetramer, typical of most other plant PEPCs studied to date. By contrast, PEPC2 appears to exist as an unusual 681- kDa hetero-octamer composed of the same p107 found in PEPC1 and an associated p64 that is structurally and immunologically unrelated to p107. Nevertheless, Q-TOF MS/MS analysis of tryptic peptides revealed that p64 is highly similar to two putative PEPCs identified by annotation of the rice and Arabidopsis genomes (Fig. 5). Although they contain conserved regions required for PEPC activity, these putative PEPCs exhibit a unique N-terminal region that lacks the regulatory seryl phosphorylation site thought to be conserved among all plant PEPCs (1Chollet R. Vidal J. O' Leary M.H. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 273-298Google Scholar) and have predicted molecular masses of 102 (rice) and 110 kDa (Arabidopsis). Although it is feasible that p64 represents an in vivo or in vitro degradation product of a larger polypeptide, the data indicate that p64 is a PEPC-like polypeptide that interacts with p107 to give rise to the COS PEPC2 heteromeric complex. This association may either physically block the allosteric sites of p107 or promote an allosteric transition in p107 such that effectors have limited access to their respective sites.COS PEPC1 and PEPC2 Emulate Green Algal 舠Class 1舡 and 舠Class 2舡 PEPC IsoformsInterestingly, the respective properties of COS PEPC1 and PEPC2 are remarkably comparable with those of the homotetrameric low Mr Class 1 and heteromeric high Mr Class 2 PEPC isoforms of the green algaeSelenastrum minutum and Chlamydomonas reinhardtii(20Rivoal J. Dunford R. Plaxton W.C. Turpin D.H. Arch. Biochem. Biophys. 1996; 332: 47-57Google Scholar, 21Rivoal J. Plaxton W.C. Turpin D.H. Biochem. J. 1998; 331: 201-209Google Scholar, 22Rivoal J. Trzos S. Gage D.A. Plaxton W.C. Turpin D.H. J. Biol. Chem. 2001; 276: 12588-12597Google Scholar). All algal PEPC isoforms share the same 102-kDa catalytic subunit (p102). Similar to COS PEPC2, the algal Class 2 PEPCs also contain associated polypeptides that are immunologically unrelated to p102. MALDI-TOF MS and microsequencing revealed that like the p64 of COS PEPC2, the 130- kDa subunit (p130) of S. minutum Class 2 PEPC represents a distinct PEPC-like polypeptide that is only distantly related to p102 (22Rivoal J. Trzos S. Gage D.A. Plaxton W.C. Turpin D.H. J. Biol. Chem. 2001; 276: 12588-12597Google Scholar). Moreover, relative to COS PEPC1 and algal Class 1 PEPCs, the COS PEPC2 and algal Class 2 PEPCs demonstrate significantly enhanced thermal stability and a much lower sensitivity to allosteric effectors and pH changes within the physiological range (20Rivoal J. Dunford R. Plaxton W.C. Turpin D.H. Arch. Biochem. Biophys. 1996; 332: 47-57Google Scholar, 21Rivoal J. Plaxton W.C. Turpin D.H. Biochem. J. 1998; 331: 201-209Google Scholar). Taken together, the data imply that high and low MrPEPC isoforms arose in green algae before the evolution of vascular plants, with this feature being conserved as a key structure-function aspect of at least some plant PEPCs.Possible Functions and Interconversion of COS PEPC1 and PEPC2Similar to PEPCs from other non-green plant tissues (6Schuller K.A. Turpin D.H. Plaxton W.C. Plant Physiol. 1990; 94: 1429-1435Google Scholar, 8Moraes T.F. Plaxton W.C. Eur. J. Biochem. 2000; 267: 4465-4476Google Scholar,9Law R.D. Plaxton W.C. Biochem. J. 1995; 307: 807-816Google Scholar, 11Podestá F.E. Plaxton W.C. Planta. 1992; 194: 381-387Google Scholar, 15Golombek S. Heim U. Horstmann C. Wobus U. Weber H. Planta. 1999; 208: 66-72Google Scholar), COS PEPC1 was activated by hexose-mono-Ps and potently inhibited by malate, Asp, and Glu at pH 7.3 (Table II). This result indicates that PEPC1 may fulfill a key anaplerotic role to replenish dicarboxylic acids consumed through transamination reactions required to support storage protein synthesis. The inhibition of PEPC1 by Asp and Glu provides a tight feedback control that could closely balance PEPC1 activity with the production of C-skeletons (i.e. oxaloacetate, 2-oxoglutarate) required for NH4+ assimilation or transamination reactions. The 舠effector-insensitive舡 PEPC2, by contrast, may facilitate PEP flux to malate in support of leucoplast fatty acid synthesis despite the significant malate levels present in developing COS (18Smith R.G. Gauthier D.A. Dennis D.T. Turpin D.H. Plant Physiol. 1992; 98: 1233-1238Google Scholar). Nondenaturing PAGE of clarified COS extracts followed by in-gel PEPC activity staining revealed that PEPC2 increases during COS development, peaking at stage VII, and then rapidly disappears during COS maturation (Fig.4C). This pattern parallels triglyceride accumulation in this tissue, which also peaks at stage VII (34Simcox P.D. Garland W. DeLuca V. Canvin D.T. Dennis D.T. Can. J. Bot. 1979; 57: 1008-1014Google Scholar). The developmental profile for PEPC1 (Fig. 4C), by contrast, parallels that of storage protein accumulation (24Greenwood J. Bewley J. Can. J. Bot. 1982; 60: 1751-1760Google Scholar), with both becoming maximal during the maturation phase of COS development. Further studies using transgenic plants and/or pharmacological inhibitors will help to fully evaluate the metabolic functions of COS PEPC1 and PEPC2.It remains to be determined whether and how COS PEPC1 and PEPC2 interconvert. However, protein-kinase-mediated phosphorylation of p102 appears to be involved in the control and structural organization of green algal (S. minutum) Class 2 PEPCs (23Rivoal J. Turpin D.H. Plaxton W.C. Plant Cell Physiol. 2002; 43: 785-792Google Scholar). COS p107 contains the N-terminal regulatory seryl phosphorylation site characteristic of most plant PEPCs (Fig. 3A). It will be of interest to determine whether COS PEPC1 and PEPC2 are interconverted via a phosphorylation-dephosphorylation mechanism involving p107 and/or p64. When partial in vitro proteolysis of p107 was prevented, two COS PEPC isoforms that significantly differed in their physical and kinetic properties were resolved by Superdex 200 FPLC and highly purified. Tissue- and/or developmentally specific PEPC isozymes are known to occur in vascular plants (1Chollet R. Vidal J. O' Leary M.H. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 273-298Google Scholar, 2Rajagopalan A., V Devi M.T. Raghavendra A.S. Photosynth. Res. 1994; 39: 115-135Google Scholar, 3Nimmo H.G. Batey N.H. Dickinson H.G. Hetherington S.M. Society for Experimental Biology Seminar Series 53: Post Translational Modifications in Plants. Cambridge University Press, Cambridge, United Kingdom1993: 161-170Google Scholar), and genetic evidence indicates that developing Glycine max (soybean) andVicia faba seeds express more than one PEPC gene (15Golombek S. Heim U. Horstmann C. Wobus U. Weber H. Planta. 1999; 208: 66-72Google Scholar, 32Sugimoto T. Kawasaki T. Kato T. Whittier R.F. Shibata D. Kawamura Y. Plant Mol. Biol. 1993; 20: 743-747Google Scholar,33Vazquez-Tello A. Whittier R.F. Kawasaki T. Sugimoto T. Kawamura Y. Shibata D. Plant Physiol. 1993; 103: 1025-1026Google Scholar). To our knowledge, this is the first report of the isolation of two PEPC isoforms from the same plant tissue. COS PEPC1 is a p107 homotetramer, typical of most other plant PEPCs studied to date. By contrast, PEPC2 appears to exist as an unusual 681- kDa hetero-octamer composed of the same p107 found in PEPC1 and an associated p64 that is structurally and immunologically unrelated to p107. Nevertheless, Q-TOF MS/MS analysis of tryptic peptides revealed that p64 is highly similar to two putative PEPCs identified by annotation of the rice and Arabidopsis genomes (Fig. 5). Although they contain conserved regions required for PEPC activity, these putative PEPCs exhibit a unique N-terminal region that lacks the regulatory seryl phosphorylation site thought to be conserved among all plant PEPCs (1Chollet R. Vidal J. O' Leary M.H. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 273-298Google Scholar) and have predicted molecular masses of 102 (rice) and 110 kDa (Arabidopsis). Although it is feasible that p64 represents an in vivo or in vitro degradation product of a larger polypeptide, the data indicate that p64 is a PEPC-like polypeptide that interacts with p107 to give rise to the COS PEPC2 heteromeric complex. This association may either physically block the allosteric sites of p107 or promote an allosteric transition in p107 such that effectors have limited access to their respective sites. COS PEPC1 and PEPC2 Emulate Green Algal 舠Class 1舡 and 舠Class 2舡 PEPC IsoformsInterestingly, the respective properties of COS PEPC1 and PEPC2 are remarkably comparable with those of the homotetrameric low Mr Class 1 and heteromeric high Mr Class 2 PEPC isoforms of the green algaeSelenastrum minutum and Chlamydomonas reinhardtii(20Rivoal J. Dunford R. Plaxton W.C. Turpin D.H. Arch. Biochem. Biophys. 1996; 332: 47-57Google Scholar, 21Rivoal J. Plaxton W.C. Turpin D.H. Biochem. J. 1998; 331: 201-209Google Scholar, 22Rivoal J. Trzos S. Gage D.A. Plaxton W.C. Turpin D.H. J. Biol. Chem. 2001; 276: 12588-12597Google Scholar). All algal PEPC isoforms share the same 102-kDa catalytic subunit (p102). Similar to COS PEPC2, the algal Class 2 PEPCs also contain associated polypeptides that are immunologically unrelated to p102. MALDI-TOF MS and microsequencing revealed that like the p64 of COS PEPC2, the 130- kDa subunit (p130) of S. minutum Class 2 PEPC represents a distinct PEPC-like polypeptide that is only distantly related to p102 (22Rivoal J. Trzos S. Gage D.A. Plaxton W.C. Turpin D.H. J. Biol. Chem. 2001; 276: 12588-12597Google Scholar). Moreover, relative to COS PEPC1 and algal Class 1 PEPCs, the COS PEPC2 and algal Class 2 PEPCs demonstrate significantly enhanced thermal stability and a much lower sensitivity to allosteric effectors and pH changes within the physiological range (20Rivoal J. Dunford R. Plaxton W.C. Turpin D.H. Arch. Biochem. Biophys. 1996; 332: 47-57Google Scholar, 21Rivoal J. Plaxton W.C. Turpin D.H. Biochem. J. 1998; 331: 201-209Google Scholar). Taken together, the data imply that high and low MrPEPC isoforms arose in green algae before the evolution of vascular plants, with this feature being conserved as a key structure-function aspect of at least some plant PEPCs.Possible Functions and Interconversion of COS PEPC1 and PEPC2Similar to PEPCs from other non-green plant tissues (6Schuller K.A. Turpin D.H. Plaxton W.C. Plant Physiol. 1990; 94: 1429-1435Google Scholar, 8Moraes T.F. Plaxton W.C. Eur. J. Biochem. 2000; 267: 4465-4476Google Scholar,9Law R.D. Plaxton W.C. Biochem. J. 1995; 307: 807-816Google Scholar, 11Podestá F.E. Plaxton W.C. Planta. 1992; 194: 381-387Google Scholar, 15Golombek S. Heim U. Horstmann C. Wobus U. Weber H. Planta. 1999; 208: 66-72Google Scholar), COS PEPC1 was activated by hexose-mono-Ps and potently inhibited by malate, Asp, and Glu at pH 7.3 (Table II). This result indicates that PEPC1 may fulfill a key anaplerotic role to replenish dicarboxylic acids consumed through transamination reactions required to support storage protein synthesis. The inhibition of PEPC1 by Asp and Glu provides a tight feedback control that could closely balance PEPC1 activity with the production of C-skeletons (i.e. oxaloacetate, 2-oxoglutarate) required for NH4+ assimilation or transamination reactions. The 舠effector-insensitive舡 PEPC2, by contrast, may facilitate PEP flux to malate in support of leucoplast fatty acid synthesis despite the significant malate levels present in developing COS (18Smith R.G. Gauthier D.A. Dennis D.T. Turpin D.H. Plant Physiol. 1992; 98: 1233-1238Google Scholar). Nondenaturing PAGE of clarified COS extracts followed by in-gel PEPC activity staining revealed that PEPC2 increases during COS development, peaking at stage VII, and then rapidly disappears during COS maturation (Fig.4C). This pattern parallels triglyceride accumulation in this tissue, which also peaks at stage VII (34Simcox P.D. Garland W. DeLuca V. Canvin D.T. Dennis D.T. Can. J. Bot. 1979; 57: 1008-1014Google Scholar). The developmental profile for PEPC1 (Fig. 4C), by contrast, parallels that of storage protein accumulation (24Greenwood J. Bewley J. Can. J. Bot. 1982; 60: 1751-1760Google Scholar), with both becoming maximal during the maturation phase of COS development. Further studies using transgenic plants and/or pharmacological inhibitors will help to fully evaluate the metabolic functions of COS PEPC1 and PEPC2.It remains to be determined whether and how COS PEPC1 and PEPC2 interconvert. However, protein-kinase-mediated phosphorylation of p102 appears to be involved in the control and structural organization of green algal (S. minutum) Class 2 PEPCs (23Rivoal J. Turpin D.H. Plaxton W.C. Plant Cell Physiol. 2002; 43: 785-792Google Scholar). COS p107 contains the N-terminal regulatory seryl phosphorylation site characteristic of most plant PEPCs (Fig. 3A). It will be of interest to determine whether COS PEPC1 and PEPC2 are interconverted via a phosphorylation-dephosphorylation mechanism involving p107 and/or p64. COS PEPC1 and PEPC2 Emulate Green Algal 舠Class 1舡 and 舠Class 2舡 PEPC IsoformsInterestingly, the respective properties of COS PEPC1 and PEPC2 are remarkably comparable with those of the homotetrameric low Mr Class 1 and heteromeric high Mr Class 2 PEPC isoforms of the green algaeSelenastrum minutum and Chlamydomonas reinhardtii(20Rivoal J. Dunford R. Plaxton W.C. Turpin D.H. Arch. Biochem. Biophys. 1996; 332: 47-57Google Scholar, 21Rivoal J. Plaxton W.C. Turpin D.H. Biochem. J. 1998; 331: 201-209Google Scholar, 22Rivoal J. Trzos S. Gage D.A. Plaxton W.C. Turpin D.H. J. Biol. Chem. 2001; 276: 12588-12597Google Scholar). All algal PEPC isoforms share the same 102-kDa catalytic subunit (p102). Similar to COS PEPC2, the algal Class 2 PEPCs also contain associated polypeptides that are immunologically unrelated to p102. MALDI-TOF MS and microsequencing revealed that like the p64 of COS PEPC2, the 130- kDa subunit (p130) of S. minutum Class 2 PEPC represents a distinct PEPC-like polypeptide that is only distantly related to p102 (22Rivoal J. Trzos S. Gage D.A. Plaxton W.C. Turpin D.H. J. Biol. Chem. 2001; 276: 12588-12597Google Scholar). Moreover, relative to COS PEPC1 and algal Class 1 PEPCs, the COS PEPC2 and algal Class 2 PEPCs demonstrate significantly enhanced thermal stability and a much lower sensitivity to allosteric effectors and pH changes within the physiological range (20Rivoal J. Dunford R. Plaxton W.C. Turpin D.H. Arch. Biochem. Biophys. 1996; 332: 47-57Google Scholar, 21Rivoal J. Plaxton W.C. Turpin D.H. Biochem. J. 1998; 331: 201-209Google Scholar). Taken together, the data imply that high and low MrPEPC isoforms arose in green algae before the evolution of vascular plants, with this feature being conserved as a key structure-function aspect of at least some plant PEPCs.Possible Functions and Interconversion of COS PEPC1 and PEPC2Similar to PEPCs from other non-green plant tissues (6Schuller K.A. Turpin D.H. Plaxton W.C. Plant Physiol. 1990; 94: 1429-1435Google Scholar, 8Moraes T.F. Plaxton W.C. Eur. J. Biochem. 2000; 267: 4465-4476Google Scholar,9Law R.D. Plaxton W.C. Biochem. J. 1995; 307: 807-816Google Scholar, 11Podestá F.E. Plaxton W.C. Planta. 1992; 194: 381-387Google Scholar, 15Golombek S. Heim U. Horstmann C. Wobus U. Weber H. Planta. 1999; 208: 66-72Google Scholar), COS PEPC1 was activated by hexose-mono-Ps and potently inhibited by malate, Asp, and Glu at pH 7.3 (Table II). This result indicates that PEPC1 may fulfill a key anaplerotic role to replenish dicarboxylic acids consumed through transamination reactions required to support storage protein synthesis. The inhibition of PEPC1 by Asp and Glu provides a tight feedback control that could closely balance PEPC1 activity with the production of C-skeletons (i.e. oxaloacetate, 2-oxoglutarate) required for NH4+ assimilation or transamination reactions. The 舠effector-insensitive舡 PEPC2, by contrast, may facilitate PEP flux to malate in support of leucoplast fatty acid synthesis despite the significant malate levels present in developing COS (18Smith R.G. Gauthier D.A. Dennis D.T. Turpin D.H. Plant Physiol. 1992; 98: 1233-1238Google Scholar). Nondenaturing PAGE of clarified COS extracts followed by in-gel PEPC activity staining revealed that PEPC2 increases during COS development, peaking at stage VII, and then rapidly disappears during COS maturation (Fig.4C). This pattern parallels triglyceride accumulation in this tissue, which also peaks at stage VII (34Simcox P.D. Garland W. DeLuca V. Canvin D.T. Dennis D.T. Can. J. Bot. 1979; 57: 1008-1014Google Scholar). The developmental profile for PEPC1 (Fig. 4C), by contrast, parallels that of storage protein accumulation (24Greenwood J. Bewley J. Can. J. Bot. 1982; 60: 1751-1760Google Scholar), with both becoming maximal during the maturation phase of COS development. Further studies using transgenic plants and/or pharmacological inhibitors will help to fully evaluate the metabolic functions of COS PEPC1 and PEPC2.It remains to be determined whether and how COS PEPC1 and PEPC2 interconvert. However, protein-kinase-mediated phosphorylation of p102 appears to be involved in the control and structural organization of green algal (S. minutum) Class 2 PEPCs (23Rivoal J. Turpin D.H. Plaxton W.C. Plant Cell Physiol. 2002; 43: 785-792Google Scholar). COS p107 contains the N-terminal regulatory seryl phosphorylation site characteristic of most plant PEPCs (Fig. 3A). It will be of interest to determine whether COS PEPC1 and PEPC2 are interconverted via a phosphorylation-dephosphorylation mechanism involving p107 and/or p64. COS PEPC1 and PEPC2 Emulate Green Algal 舠Class 1舡 and 舠Class 2舡 PEPC IsoformsInterestingly, the respective properties of COS PEPC1 and PEPC2 are remarkably comparable with those of the homotetrameric low Mr Class 1 and heteromeric high Mr Class 2 PEPC isoforms of the green algaeSelenastrum minutum and Chlamydomonas reinhardtii(20Rivoal J. Dunford R. Plaxton W.C. Turpin D.H. Arch. Biochem. Biophys. 1996; 332: 47-57Google Scholar, 21Rivoal J. Plaxton W.C. Turpin D.H. Biochem. J. 1998; 331: 201-209Google Scholar, 22Rivoal J. Trzos S. Gage D.A. Plaxton W.C. Turpin D.H. J. Biol. Chem. 2001; 276: 12588-12597Google Scholar). All algal PEPC isoforms share the same 102-kDa catalytic subunit (p102). Similar to COS PEPC2, the algal Class 2 PEPCs also contain associated polypeptides that are immunologically unrelated to p102. MALDI-TOF MS and microsequencing revealed that like the p64 of COS PEPC2, the 130- kDa subunit (p130) of S. minutum Class 2 PEPC represents a distinct PEPC-like polypeptide that is only distantly related to p102 (22Rivoal J. Trzos S. Gage D.A. Plaxton W.C. Turpin D.H. J. Biol. Chem. 2001; 276: 12588-12597Google Scholar). Moreover, relative to COS PEPC1 and algal Class 1 PEPCs, the COS PEPC2 and algal Class 2 PEPCs demonstrate significantly enhanced thermal stability and a much lower sensitivity to allosteric effectors and pH changes within the physiological range (20Rivoal J. Dunford R. Plaxton W.C. Turpin D.H. Arch. Biochem. Biophys. 1996; 332: 47-57Google Scholar, 21Rivoal J. Plaxton W.C. Turpin D.H. Biochem. J. 1998; 331: 201-209Google Scholar). Taken together, the data imply that high and low MrPEPC isoforms arose in green algae before the evolution of vascular plants, with this feature being conserved as a key structure-function aspect of at least some plant PEPCs. Interestingly, the respective properties of COS PEPC1 and PEPC2 are remarkably comparable with those of the homotetrameric low Mr Class 1 and heteromeric high Mr Class 2 PEPC isoforms of the green algaeSelenastrum minutum and Chlamydomonas reinhardtii(20Rivoal J. Dunford R. Plaxton W.C. Turpin D.H. Arch. Biochem. Biophys. 1996; 332: 47-57Google Scholar, 21Rivoal J. Plaxton W.C. Turpin D.H. Biochem. J. 1998; 331: 201-209Google Scholar, 22Rivoal J. Trzos S. Gage D.A. Plaxton W.C. Turpin D.H. J. Biol. Chem. 2001; 276: 12588-12597Google Scholar). All algal PEPC isoforms share the same 102-kDa catalytic subunit (p102). Similar to COS PEPC2, the algal Class 2 PEPCs also contain associated polypeptides that are immunologically unrelated to p102. MALDI-TOF MS and microsequencing revealed that like the p64 of COS PEPC2, the 130- kDa subunit (p130) of S. minutum Class 2 PEPC represents a distinct PEPC-like polypeptide that is only distantly related to p102 (22Rivoal J. Trzos S. Gage D.A. Plaxton W.C. Turpin D.H. J. Biol. Chem. 2001; 276: 12588-12597Google Scholar). Moreover, relative to COS PEPC1 and algal Class 1 PEPCs, the COS PEPC2 and algal Class 2 PEPCs demonstrate significantly enhanced thermal stability and a much lower sensitivity to allosteric effectors and pH changes within the physiological range (20Rivoal J. Dunford R. Plaxton W.C. Turpin D.H. Arch. Biochem. Biophys. 1996; 332: 47-57Google Scholar, 21Rivoal J. Plaxton W.C. Turpin D.H. Biochem. J. 1998; 331: 201-209Google Scholar). Taken together, the data imply that high and low MrPEPC isoforms arose in green algae before the evolution of vascular plants, with this feature being conserved as a key structure-function aspect of at least some plant PEPCs. Possible Functions and Interconversion of COS PEPC1 and PEPC2Similar to PEPCs from other non-green plant tissues (6Schuller K.A. Turpin D.H. Plaxton W.C. Plant Physiol. 1990; 94: 1429-1435Google Scholar, 8Moraes T.F. Plaxton W.C. Eur. J. Biochem. 2000; 267: 4465-4476Google Scholar,9Law R.D. Plaxton W.C. Biochem. J. 1995; 307: 807-816Google Scholar, 11Podestá F.E. Plaxton W.C. Planta. 1992; 194: 381-387Google Scholar, 15Golombek S. Heim U. Horstmann C. Wobus U. Weber H. Planta. 1999; 208: 66-72Google Scholar), COS PEPC1 was activated by hexose-mono-Ps and potently inhibited by malate, Asp, and Glu at pH 7.3 (Table II). This result indicates that PEPC1 may fulfill a key anaplerotic role to replenish dicarboxylic acids consumed through transamination reactions required to support storage protein synthesis. The inhibition of PEPC1 by Asp and Glu provides a tight feedback control that could closely balance PEPC1 activity with the production of C-skeletons (i.e. oxaloacetate, 2-oxoglutarate) required for NH4+ assimilation or transamination reactions. The 舠effector-insensitive舡 PEPC2, by contrast, may facilitate PEP flux to malate in support of leucoplast fatty acid synthesis despite the significant malate levels present in developing COS (18Smith R.G. Gauthier D.A. Dennis D.T. Turpin D.H. Plant Physiol. 1992; 98: 1233-1238Google Scholar). Nondenaturing PAGE of clarified COS extracts followed by in-gel PEPC activity staining revealed that PEPC2 increases during COS development, peaking at stage VII, and then rapidly disappears during COS maturation (Fig.4C). This pattern parallels triglyceride accumulation in this tissue, which also peaks at stage VII (34Simcox P.D. Garland W. DeLuca V. Canvin D.T. Dennis D.T. Can. J. Bot. 1979; 57: 1008-1014Google Scholar). The developmental profile for PEPC1 (Fig. 4C), by contrast, parallels that of storage protein accumulation (24Greenwood J. Bewley J. Can. J. Bot. 1982; 60: 1751-1760Google Scholar), with both becoming maximal during the maturation phase of COS development. Further studies using transgenic plants and/or pharmacological inhibitors will help to fully evaluate the metabolic functions of COS PEPC1 and PEPC2.It remains to be determined whether and how COS PEPC1 and PEPC2 interconvert. However, protein-kinase-mediated phosphorylation of p102 appears to be involved in the control and structural organization of green algal (S. minutum) Class 2 PEPCs (23Rivoal J. Turpin D.H. Plaxton W.C. Plant Cell Physiol. 2002; 43: 785-792Google Scholar). COS p107 contains the N-terminal regulatory seryl phosphorylation site characteristic of most plant PEPCs (Fig. 3A). It will be of interest to determine whether COS PEPC1 and PEPC2 are interconverted via a phosphorylation-dephosphorylation mechanism involving p107 and/or p64. Similar to PEPCs from other non-green plant tissues (6Schuller K.A. Turpin D.H. Plaxton W.C. Plant Physiol. 1990; 94: 1429-1435Google Scholar, 8Moraes T.F. Plaxton W.C. Eur. J. Biochem. 2000; 267: 4465-4476Google Scholar,9Law R.D. Plaxton W.C. Biochem. J. 1995; 307: 807-816Google Scholar, 11Podestá F.E. Plaxton W.C. Planta. 1992; 194: 381-387Google Scholar, 15Golombek S. Heim U. Horstmann C. Wobus U. Weber H. Planta. 1999; 208: 66-72Google Scholar), COS PEPC1 was activated by hexose-mono-Ps and potently inhibited by malate, Asp, and Glu at pH 7.3 (Table II). This result indicates that PEPC1 may fulfill a key anaplerotic role to replenish dicarboxylic acids consumed through transamination reactions required to support storage protein synthesis. The inhibition of PEPC1 by Asp and Glu provides a tight feedback control that could closely balance PEPC1 activity with the production of C-skeletons (i.e. oxaloacetate, 2-oxoglutarate) required for NH4+ assimilation or transamination reactions. The 舠effector-insensitive舡 PEPC2, by contrast, may facilitate PEP flux to malate in support of leucoplast fatty acid synthesis despite the significant malate levels present in developing COS (18Smith R.G. Gauthier D.A. Dennis D.T. Turpin D.H. Plant Physiol. 1992; 98: 1233-1238Google Scholar). Nondenaturing PAGE of clarified COS extracts followed by in-gel PEPC activity staining revealed that PEPC2 increases during COS development, peaking at stage VII, and then rapidly disappears during COS maturation (Fig.4C). This pattern parallels triglyceride accumulation in this tissue, which also peaks at stage VII (34Simcox P.D. Garland W. DeLuca V. Canvin D.T. Dennis D.T. Can. J. Bot. 1979; 57: 1008-1014Google Scholar). The developmental profile for PEPC1 (Fig. 4C), by contrast, parallels that of storage protein accumulation (24Greenwood J. Bewley J. Can. J. Bot. 1982; 60: 1751-1760Google Scholar), with both becoming maximal during the maturation phase of COS development. Further studies using transgenic plants and/or pharmacological inhibitors will help to fully evaluate the metabolic functions of COS PEPC1 and PEPC2. It remains to be determined whether and how COS PEPC1 and PEPC2 interconvert. However, protein-kinase-mediated phosphorylation of p102 appears to be involved in the control and structural organization of green algal (S. minutum) Class 2 PEPCs (23Rivoal J. Turpin D.H. Plaxton W.C. Plant Cell Physiol. 2002; 43: 785-792Google Scholar). COS p107 contains the N-terminal regulatory seryl phosphorylation site characteristic of most plant PEPCs (Fig. 3A). It will be of interest to determine whether COS PEPC1 and PEPC2 are interconverted via a phosphorylation-dephosphorylation mechanism involving p107 and/or p64. We thank Dr. Jean Rivoal (University of Montreal) for helpful discussions. We also gratefully acknowledge Dr. David Hyndman of the Queen's Protein Function Discovery Research and Training Program for his invaluable assistance with the MS analyses of p107 and p64.