Regulatory Monoubiquitination of Phosphoenolpyruvate Carboxylase in Germinating Castor Oil Seeds

Abstract

Phosphoenolpyruvate carboxylase (PEPC) is a tightly regulated enzyme situated at the core of plant C-metabolism. Although its anaplerotic role and control by allosteric effectors, reversible phosphorylation, and oligomerization have been well documented in the endosperm of developing castor oil seeds (COS), relatively little is known about PEPC in germinating COS. The initial phase of COS germination was accompanied by elevated PEPC activity and accumulation of comparable amounts of pre-existing 107-kDa and inducible 110-kDa immunoreactive PEPC polypeptides (p107 and p110, respectively). A 440-kDa PEPC heterotetramer composed of an equivalent ratio of nonphosphorylated p110 and p107 subunits was purified from germinated COS. N-terminal microsequencing, mass spectrometry, and immunoblotting revealed that both subunits arose from the same gene (RcPpc3) that encodes the p107 subunit of a phosphorylated 410-kDa PEPC homotetramer in developing COS but that p110 is a monoubiquitinated form of p107. Tandem mass spectrometry sequencing of a diglycinated tryptic peptide identified Lys-628 as p110's monoubiquitination site. This residue is conserved in vascular plant PEPCs and is proximal to a PEP-binding/catalytic domain. Incubation with a human deubiquitinating enzyme (USP-2 core) converted the p110:p107 PEPC heterotetramer into a p107 homotetramer while significantly reducing the enzyme's K m (PEP) and sensitivity to allosteric activators (hexose-Ps, glycerol-3-P) and inhibitors (malate, aspartate). Monoubiquitination is a non-destructive and reversible post-translational modification involved in the control of diverse processes such as transcription, endocytosis, and signal transduction. The current study demonstrates that tissue-specific monoubiquitination of a metabolic enzyme can also occur and that this modification influences its kinetic and regulatory properties.

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

“…Class-1 PEPCs also fulfill a wide range of crucial nonphotosynthetic functions, particularly the anaplerotic replenishment of tricarboxylic acid cycle intermediates consumed during biosynthesis (O'Leary et al, 2011a). Class-1 PEPCs are subject to a complex set of posttranslational controls including allosteric effectors, covalent modification via phosphorylation or monoubiquitination, and protein-protein interactions (Uhrig et al, 2008;O'Leary et al, 2009O'Leary et al, , 2011aO'Leary et al, , 2011b.…”

“…Phosphorylation at a conserved N-terminal seryl residue activates Class-1 PEPCs by decreasing inhibition by malate while increasing activation by Glc-6-P. By contrast, Class-1 PEPC is subject to inhibitory monoubiquitination during castor oil (Ricinus communis) seed (COS) germination, or following depodding of developing COS (Uhrig et al, 2008;O'Leary et al, 2011b). Immunoblots of germinating COS extracts revealed a 1:1 ratio of immunoreactive 110-and 107-kD PTPC polypeptides (p110 and p107, respectively).…”

“…Immunoblots of germinating COS extracts revealed a 1:1 ratio of immunoreactive 110-and 107-kD PTPC polypeptides (p110 and p107, respectively). PEPC purification and mass spectrometry (MS) demonstrated that (1) p110 and p107 are subunits of a 440-kD Class-1 PEPC heterotetramer, (2) both subunits arise from the same PTPC gene (RcPpc3) that also encodes the phosphorylated 410-kD Class-1 PEPC homotetramer of intact developing COS, and (3) p110 is a monoubiquitinated form of p107 (Uhrig et al, 2008). The monoubiquitination site (Lys-628) of COS p110 is conserved in vascular plant PEPCs and is proximal to a PEP-binding/ catalytic domain.…”

“…The monoubiquitination site (Lys-628) of COS p110 is conserved in vascular plant PEPCs and is proximal to a PEP-binding/ catalytic domain. Incubation with a deubiquitinating enzyme converted the Class-1 PEPC p110:p107 heterotetramer into a p107 homotetramer while exerting significant effects on the enzyme's kinetic properties (Uhrig et al, 2008). PTPC monoubiquitination rather than phosphorylation is widespread throughout the astor plant and appears to be the predominant posttranslational modification (PTM) of Class-1 PEPC that occurs in unstressed plants (O'Leary et al, 2011b).…”

Light-dependent activation of phosphoenolpyruvate carboxylase by reversible phosphorylation in cluster roots of white lupin plants: diurnal control in response to photosynthate supply

“…Different letters denote significant differences (P < 0Á05) from the respective initial, 8L value. The lanes of the anti-PEPC and anti-pSer11 blots labelled 'AtPEPC' contained 50 and 100 ng, respectively, of purified phosphorylated AtPPC1 from ÀPi arabidopsis suspension cells (Gregory et al, 2009), whereas those labelled 'Castor PEPC' contained 50 and 250 ng, respectively, of dephosphorylated, partially monoubiquitinated PEPC purified from germinating castor beans (Uhrig et al, 2008). 0Á28 6 0Á01 0Á15 6 0Á02 À1Á8 6 0Á2 † Citrate 24Á5 6 0Á6 2 2 Á9 6 0Á9 À1Á1 6 0Á02 Malate 10Á2 6 0Á3 6 Á5 6 0Á3 À1Á5 6 0Á04 † nmol g À1 fresh weight Sucrose-6-phosphate 0Á063 6 0Á004 0Á015 6 0Á001 À4Á2 6 0Á3 † Trehalose-6-phosphate 0Á016 6 0Á001 0Á004 6 0Á0003 À4Á0 6 0Á3 † Galactose-1-phosphate 1Á1 6 0Á1 0 Á56 6 0Á03 À1Á9 6 0Á2 † Glucose-1-phosphate 2Á8 6 0Á3 1 Á2 6 0Á1 À2Á3 6 0Á3 † UDP-glucose 8Á6 6 1Á4 3 Á8 6 0Á5 À2Á2 6 0Á4 † ADP-glucose 0Á027 6 0Á003 0Á023 6 0Á001 À1Á1 6 0Á1 Glycerol-3-phosphate 0Á24 6 0Á02 0Á20 6 0Á01 À1Á2 6 0Á1 Glycerate 60 6 3Á7 4 7 6 1Á9 À1Á2 6 0Á1 Pyruvate 53Á6 6 1Á8 3 4 Á7 6 1Á8 À1Á5 6 0Á1 † Shikimate 7Á3 6 0Á30 4Á0 6 0Á2 À1Á8 6 0Á1 † Isocitrate 162 6 5 144 6 7 À1Á1 6 0Á03 Aconitate 75Á1 6 2Á5 6 6 Á7 6 3Á6 À1Á1 6 0Á04 2-Oxoglutarate 57Á6 6 1Á7 2 3 Á0 6 1Á1 À2Á5 6 0Á1 † Succinate 168 6 5 103 6 4 À1Á6 6 0Á04 † Fumarate 126 6 3 111 6 4 À1Á1 6 0Á02…”