Abstract
Accumulating evidence indicates important functions for phosphoenolpyruvate (PEP) carboxylase (PEPC) in inorganic phosphate (Pi)-starved plants. This includes controlling the production of organic acid anions (malate, citrate) that are excreted in copious amounts by proteoid roots of nonmycorrhizal species such as harsh hakea (Hakea prostrata). This, in turn, enhances the bioavailability of mineral-bound Pi by solubilizing Al(3+), Fe(3+), and Ca(2+) phosphates in the rhizosphere. Harsh hakea thrives in the nutrient-impoverished, ancient soils of southwestern Australia. Proteoid roots from Pi-starved harsh hakea were analyzed over 20 d of development to correlate changes in malate and citrate exudation with PEPC activity, posttranslational modifications (inhibitory monoubiquitination versus activatory phosphorylation), and kinetic/allosteric properties. Immature proteoid roots contained an equivalent ratio of monoubiquitinated 110-kD and phosphorylated 107-kD PEPC polypeptides (p110 and p107, respectively). PEPC purification, immunoblotting, and mass spectrometry indicated that p110 and p107 are subunits of a 430-kD heterotetramer and that they both originate from the same plant-type PEPC gene. Incubation with a deubiquitinating enzyme converted the p110:p107 PEPC heterotetramer of immature proteoid roots into a p107 homotetramer while significantly increasing the enzyme's activity under suboptimal but physiologically relevant assay conditions. Proteoid root maturation was paralleled by PEPC activation (e.g. reduced Km [PEP] coupled with elevated I50 [malate and Asp] values) via in vivo deubiquitination of p110 to p107, and subsequent phosphorylation of the deubiquitinated subunits. This novel mechanism of posttranslational control is hypothesized to contribute to the massive synthesis and excretion of organic acid anions that dominates the carbon metabolism of the mature proteoid roots.
Highlights
IntroductionPEPC catalyzes the irreversible β-carboxylation of phosphoenolpyruvate (PEP) to form oxaloacetate (OAA) and Pi. Vascular plant PEPCs belong to a small multigene family encoding several closely related plant-type PEPCs (PTPCs), along with a distantly related bacterial-type
Phosphoenolpyruvate carboxylase (PEPC) (EC 4.1.1.31) is a ubiquitous and tightly regulated cytosolic enzyme of vascular plants that is widely distributed in green algae and bacteria.PEPC catalyzes the irreversible β-carboxylation of phosphoenolpyruvate (PEP) to form oxaloacetate (OAA) and Pi
BTPC’s tight interaction with co-expressed plant-type PEPCs (PTPCs) subunits. This association results in the formation of unusual ~900-kD Class-2 PEPC hetero-octameric complexes that are largely desensitized to allosteric effectors and that dynamically associate with the surface of mitochondria in vivo (O’Leary et al, 2009, 2011a; Igawa et al, 2010; Park et al, 2012)
Summary
PEPC catalyzes the irreversible β-carboxylation of phosphoenolpyruvate (PEP) to form oxaloacetate (OAA) and Pi. Vascular plant PEPCs belong to a small multigene family encoding several closely related plant-type PEPCs (PTPCs), along with a distantly related bacterial-type. PTPC genes encode 105 to 110-kD polypeptides that typically assemble as ~400-kD Class-1 PEPC homotetramers. BTPC genes encode larger 116- to118-kD polypeptides owing to a unique intrinsically disordered region that mediates. BTPC’s tight interaction with co-expressed PTPC subunits. This association results in the formation of unusual ~900-kD Class-2 PEPC hetero-octameric complexes that are largely desensitized to allosteric effectors and that dynamically associate with the surface of mitochondria in vivo (O’Leary et al, 2009, 2011a; Igawa et al, 2010; Park et al, 2012)
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