Regulatory monoubiquitination of phosphoenolpyruvate carboxylase in germinating castor oil seeds.
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 non-phosphorylated 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.
Uhrig
R Glen
RG
Department of Biology, Queen's University, Kingston, Ontario K7L 3N6, Canada.
She
Yi-Min
YM
Leach
Craig A
CA
Plaxton
William C
WC
D000495:Allosteric Site; D000595:Amino Acid Sequence; D002368:Castor Oil / Q000737:chemistry*; D005786:Gene Expression Regulation; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010730:Phosphoenolpyruvate Carboxylase / Q000737:chemistry* / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry; D017434:Protein Structure, Tertiary; D012639:Seeds / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D025801:Ubiquitin / Q000378:metabolism*
D010940:Plant Proteins; D025801:Ubiquitin; D002368:Castor Oil; D010730:Phosphoenolpyruvate Carboxylase