Structural and kinetic properties of high and low molecular mass phosphoenolpyruvate carboxylase isoforms from the endosperm of developing castor oilseeds

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 low M(r) Class 1 and heteromeric high M(r) Class 2 PEPC isoforms of unicellular green algae.     

The bacterial-type phosphoenolpyruvate carboxylase isozyme from developing castor oil seeds is subject to in vivo regulatory phosphorylation at serine-451

Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled anaplerotic enzyme situated at a pivotal branch point of plant carbohydrate-metabolism. In developing castor oil seeds (COS) a novel allosterically-densensitized 910-kDa Class-2 PEPC hetero-octameric complex arises from a tight interaction between 107-kDa plant-type PEPC and 118-kDa bacterial-type PEPC (BTPC) subunits. Mass spectrometry and immunoblotting with anti-phosphoSer451 specific antibodies established that COS BTPC is in vivo phosphorylated at Ser451, a highly conserved target residue that occurs within an intrinsically disordered region. This phosphorylation was enhanced during COS development or in response to depodding. Kinetic characterization of a phosphomimetic (S451D) mutant indicated that Ser451 phosphorylation inhibits the catalytic activity of BTPC subunits within the Class-2 PEPC complex.     

Bacterial- and plant-type phosphoenolpyruvate carboxylase isozymes from developing castor oil seeds interact in vivo and associate with the surface of mitochondria

Phosphoenolpyruvate carboxylase (PEPC) from developing castor oil seeds (COS) exists as two distinct oligomeric isoforms. The typical class-1 PEPC homotetramer consists of 107-kDa plant-type PEPC (PTPC) subunits, whereas the allosterically desensitized 910-kDa class-2 PEPC hetero-octamer arises from the association of class-1 PEPC with 118-kDa bacterial-type PEPC (BTPC) subunits. The in vivo interaction and subcellular location of COS BTPC and PTPC were assessed by imaging fluorescent protein (FP)-tagged PEPCs in tobacco suspension-cultured cells. The BTPC-FP mainly localized to cytoplasmic punctate/globular structures, identified as mitochondria by co-immunostaining of endogenous cytochrome oxidase. Inhibition of respiration with KCN resulted in proportional decreases and increases in mitochondrial versus cytosolic BTPC-FP, respectively. The FP-PTPC and NLS-FP-PTPC (containing an appended nuclear localization signal, NLS) localized to the cytosol and nucleus, respectively, but both co-localized with mitochondrial-associated BTPC when co-expressed with BTPC-FP. Transmission electron microscopy of immunogold-labeled developing COS revealed that BTPC and PTPC are localized at the mitochondrial (outer) envelope, as well as the cytosol. Moreover, thermolysin-sensitive BTPC and PTPC polypeptides were detected on immunoblots of purified COS mitochondria. Overall, our results demonstrate that: (i) COS BTPC and PTPC interact in vivo as a class-2 PEPC complex that associates with the surface of mitochondria, (ii) BTPC's unique and divergent intrinsically disordered region mediates its interaction with PTPC, whereas (iii) the PTPC-containing class-1 PEPC is entirely cytosolic. We hypothesize that mitochondrial-associated class-2 PEPC facilitates rapid refixation of respiratory CO(2) while sustaining a large anaplerotic flux to replenish tricarboxylic acid cycle C-skeletons withdrawn for biosynthesis.     

Phosphorylation of bacterial-type phosphoenolpyruvate carboxylase at Ser425 provides a further tier of enzyme control in developing castor oil seeds

PEPC [PEP (phosphoenolpyruvate) carboxylase] is a tightly controlled anaplerotic enzyme situated at a pivotal branch point of plant carbohydrate metabolism. Two distinct oligomeric PEPC classes were discovered in developing COS (castor oil seeds). Class-1 PEPC is a typical homotetramer of 107?kDa PTPC (plant-type PEPC) subunits, whereas the novel 910-kDa Class-2 PEPC hetero-octamer arises from a tight interaction between Class-1 PEPC and 118?kDa BTPC (bacterial-type PEPC) subunits. Mass spectrometric analysis of immunopurified COS BTPC indicated that it is subject to in vivo proline-directed phosphorylation at Ser425. We show that immunoblots probed with phosphorylation site-specific antibodies demonstrated that Ser425 phosphorylation is promoted during COS development, becoming maximal at stage IX (maturation phase) or in response to depodding. Kinetic analyses of a recombinant, chimaeric Class-2 PEPC containing phosphomimetic BTPC mutant subunits (S425D) indicated that Ser425 phosphorylation results in significant BTPC inhibition by: (i) increasing its Km(PEP) 3-fold, (ii) reducing its I50 (L-malate and L-aspartate) values by 4.5- and 2.5-fold respectively, while (iii) decreasing its activity within the physiological pH range. The developmental pattern and kinetic influence of Ser425 BTPC phosphorylation is very distinct from the in vivo phosphorylation/activation of COS Class-1 PEPC's PTPC subunits at Ser11. Collectively, the results establish that BTPC's phospho-Ser425 content depends upon COS developmental and physiological status and that Ser425 phosphorylation attenuates the catalytic activity of BTPC subunits within a Class-2 PEPC complex. To the best of our knowledge, this study provides the first evidence for protein phosphorylation as a mechanism for the in vivo control of vascular plant BTPC activity.     

In vivo regulatory phosphorylation of novel phosphoenolpyruvate carboxylase isoforms in endosperm of developing castor oil seeds

Our previous research characterized two phosphoenolpyruvate (PEP) carboxylase (PEPC) isoforms (PEPC1 and PEPC2) from developing castor oil seeds (COS). The association of a shared 107-kD subunit (p107) with an immunologically unrelated bacterial PEPC-type 64-kD polypeptide (p64) leads to marked physical and kinetic differences between the PEPC1 p107 homotetramer and PEPC2 p107/p64 heterooctamer. Here, we describe the production of antiphosphorylation site-specific antibodies to the conserved p107 N-terminal serine-6 phosphorylation site. Immunoblotting established that the serine-6 of p107 is phosphorylated in COS PEPC1 and PEPC2. This phosphorylation was reversed in vitro following incubation of clarified COS extracts or purified PEPC1 or PEPC2 with mammalian protein phosphatase type 2A and is not involved in a potential PEPC1 and PEPC2 interconversion. Similar to other plant PEPCs examined to date, p107 phosphorylation increased PEPC1 activity at pH 7.3 by decreasing its K(m)(PEP) and sensitivity to L-malate inhibition, while enhancing glucose-6-P activation. By contrast, p107 phosphorylation increased PEPC2's K(m)(PEP) and sensitivity to malate, glutamic acid, and aspartic acid inhibition. Phosphorylation of p107 was promoted during COS development (coincident with a >5-fold increase in the I(50) [malate] value for total PEPC activity in desalted extracts) but disappeared during COS desiccation. The p107 of stage VII COS became fully dephosphorylated in planta 48 h following excision of COS pods or following 72 h of dark treatment of intact plants. The in vivo phosphorylation status of p107 appears to be modulated by photosynthate recently translocated from source leaves into developing COS.     

Coimmunopurification of phosphorylated bacterial- and plant-type phosphoenolpyruvate carboxylases with the plastidial pyruvate dehydrogenase complex from developing castor oil seeds

The phosphoenolpyruvate carboxylase (PEPC) interactome of developing castor oil seed (COS; Ricinus communis) endosperm was assessed using coimmunopurification (co-IP) followed by proteomic analysis. Earlier studies suggested that immunologically unrelated 107-kD plant-type PEPCs (p107/PTPC) and 118-kD bacterial-type PEPCs (p118/BTPC) are subunits of an unusual 910-kD hetero-octameric class 2 PEPC complex of developing COS. The current results confirm that a tight physical interaction occurs between p118 and p107 because p118 quantitatively coimmunopurified with p107 following elution of COS extracts through an anti-p107-IgG immunoaffinity column. No PEPC activity or immunoreactive PEPC polypeptides were detected in the corresponding flow-through fractions. Although BTPCs lack the N-terminal phosphorylation motif characteristic of PTPCs, Pro-Q Diamond phosphoprotein staining, immunoblotting with phospho-serine (Ser)/threonine Akt substrate IgG, and phosphate-affinity PAGE established that coimmunopurified p118 was multiphosphorylated at unique Ser and/or threonine residues. Tandem mass spectrometric analysis of an endoproteinase Lys-C p118 peptide digest demonstrated that Ser-425 is subject to in vivo proline-directed phosphorylation. The co-IP of p118 with p107 did not appear to be influenced by their phosphorylation status. Because p118 phosphorylation was unchanged 48 h following elimination of photosynthate supply due to COS depodding, the signaling mechanisms responsible for photosynthate-dependent p107 phosphorylation differ from those controlling p118's in vivo phosphorylation. A 110-kD PTPC coimmunopurified with p118 and p107 when depodded COS was used. The plastidial pyruvate dehydrogenase complex (PDC(pl)) was identified as a novel PEPC interactor. Thus, a putative metabolon involving PEPC and PDC(pl) could function to channel carbon from phosphoenolpyruvate to acetyl-coenzyme A and/or to recycle CO(2) from PDC(pl) to PEPC.     

In vitro proteolysis of phosphoenolpyruvate carboxylase from developing castor oil seeds by an endogenous thiol endopeptidase

Two novel phosphoenolpyruvate carboxylase (PEPC) isoforms have been biochemically characterized from endosperm of developing castor oil seeds (COS). The association of a 107 kDa PEPC subunit (p107) with an immunologically unrelated bacterial PEPC-type 64 kDa polypeptide leads to marked physical and kinetic differences between the PEPC1 p107 homotetramer and PEPC2 p107/p64 heterooctamer. COS p107 is quite susceptible to limited proteolysis during PEPC purification. An endogenous asparaginyl endopeptidase appears to catalyze the in vitro cleavage of an approximately 120 amino acid polypeptide from the N-terminal end of p107, producing a truncated 98 kDa polypeptide (p98). Immunoblotting was used to estimate proteolytic activity by following the disappearance of p107 and concomitant appearance of p98 during incubation of clarified COS extracts at 4 degrees C. The in vitro proteolysis of p107 to p98 only occurred in the combined presence of 2 mM dithiothreitol and high salt concentrations (particularly SO(4) (2-) and PO(4) (2-) salts). Although p107-degrading activity was present throughout COS development, it was most pronounced in endosperm extracts from older beans. Several protease inhibitors, including two commercially available protease inhibitor cocktails, were tested for their ability to prevent p107 proteolysis. All of the inhibitors were ineffective except for 2,2'-dipyridyl disulfide (DPDS), a relatively inexpensive and underutilized active site inhibitor of plant thiol proteases. Asparaginyl endopeptidase activity of COS extracts was unaffected by 20% (NH(4))(2)SO(4) when determined in the presence or absence of 2 mM dithiothreitol using a spectrophotometric assay based upon the hydrolysis of benzoyl-L-Asn-p-nitroanilide. Thus, we propose that the combined presence of 2 mM dithiothreitol and 20% (NH(4))(2)SO(4) promotes a p107 conformational change that exposes the N-terminal region asparaginyl residue where p107 hydrolysis is believed to occur.     

The remarkable diversity of plant PEPC (phosphoenolpyruvate carboxylase): recent insights into the physiological functions and post-translational controls of non-photosynthetic PEPCs

PEPC [PEP (phosphoenolpyruvate) carboxylase] is a tightly controlled enzyme located at the core of plant C-metabolism that catalyses the irreversible β-carboxylation of PEP to form oxaloacetate and Pi. The critical role of PEPC in assimilating atmospheric CO(2) during C(4) and Crassulacean acid metabolism photosynthesis has been studied extensively. PEPC also fulfils a broad spectrum of non-photosynthetic functions, particularly the anaplerotic replenishment of tricarboxylic acid cycle intermediates consumed during biosynthesis and nitrogen assimilation. An impressive array of strategies has evolved to co-ordinate in vivo PEPC activity with cellular demands for C(4)-C(6) carboxylic acids. To achieve its diverse roles and complex regulation, PEPC belongs to a small multigene family encoding several closely related PTPCs (plant-type PEPCs), along with a distantly related BTPC (bacterial-type PEPC). PTPC genes encode ~110-kDa polypeptides containing conserved serine-phosphorylation and lysine-mono-ubiquitination sites, and typically exist as homotetrameric Class-1 PEPCs. In contrast, BTPC genes encode larger ~117-kDa polypeptides owing to a unique intrinsically disordered domain that mediates BTPC's tight interaction with co-expressed PTPC subunits. This association results in the formation of unusual ~900-kDa Class-2 PEPC hetero-octameric complexes that are desensitized to allosteric effectors. BTPC is a catalytic and regulatory subunit of Class-2 PEPC that is subject to multi-site regulatory phosphorylation in vivo. The interaction between divergent PEPC polypeptides within Class-2 PEPCs adds another layer of complexity to the evolution, physiological functions and metabolic control of this essential CO(2)-fixing plant enzyme. The present review summarizes exciting developments concerning the functions, post-translational controls and subcellular location of plant PTPC and BTPC isoenzymes.     

Phospho<Emphasis Type="Italic">enol</Emphasis>pyruvate carboxylase protein kinase from developing castor oil seeds: partial purification,characterization, and reversible control by photosynthate supply

Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) protein kinase (PPCK) was purified ∼1,500-fold from developing castor oil seeds (COS). Gel filtration and immunoblotting with anti-(rice PPCK2)-immune serum indicated that this Ca²⁺-insensitive PPCK exists as a 31-kDa monomer. COS PPCK-mediated rephosphorylation of the 107-kDa subunit (p107) of COS PEPC1 (K _m = 2.2 μM) activated PEPC1 by ∼80% when assayed under suboptimal conditions (pH 7.3, 0.2 mM PEP, and 0.125 mM malate). COS PPCK displayed remarkable selectivity for phosphorylating COS PEPC1 (relative to tobacco, sorghum, or maize PEPCs), exhibited a broad pH-activity optima of ∼pH 8.5, and at pH 7.3 was activated 40–65% by 1 mM PEP, or 10 mM Gln or Asn, but inhibited 65% by 10 mM L-malate. The possible control of COS PPCK by disulfide-dithiol interconversion was suggested by its rapid inactivation and subsequent reactivation when incubated with oxidized glutathione and then dithiothreitol. In vitro PPCK activity correlated with in vivo p107 phosphorylation status, with both peaking in mid-cotyledon to full-cotyledon developing COS. Notably, PPCK activity and p107 phosphorylation of developing COS were eliminated following pod excision or prolonged darkness of intact plants. Both effects were fully reversed 12 h following reillumination of darkened plants. These results implicate a direct relationship between the up-regulation of COS PPCK and p107 phosphorylation during the recommencement of photosynthate delivery from illuminated leaves to the non-photosynthetic COS. Overall, the results support the hypothesis that PEPC and PPCK participate in the control of photosynthate partitioning into C-skeletons needed as precursors for key biosynthetic pathways of developing COS. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Partial purification and biochemical characterization of a heteromeric protein phosphatase 2A holoenzyme from maize (Zea mays L.) leaves that dephosphorylates C4 phosophoenolpyruvate carboxylase

The activity and allosteric properties of plant phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) are controlled posttranslationally by specific reversible phosphorylation of a strictly conserved serine residue near the N-terminus. This up/down-regulation of PEPC is catalyzed by a dedicated and highly regulated serine/threonine (Ser/Thr) kinase (PEPC-kinase) and an opposing type-2A Ser/Thr phosphatase (PP2A). In marked contrast to PEPC-kinase, the PP2A holoenzyme from photosynthetic tissue has been virtually unstudied to date. In the present investigation, we have partially purified and characterized the native form of this PP2A from illuminated leaves of maize (Zea mays L.), a C4 plant, using maize [32P]PEPC as substrate. Various conventional chromatographic matrices, together with thiophosphorylated C4 PEPC-peptide and microcystin-LR affinity-supports, were exploited for the enrichment of this PP2A from soluble leaf extracts. Biochemical and immunological results indicate that the C4-leaf holoenzyme is analogous to other eukaryotic PP2As in being a approximately 170-kDa heteromer comprised of a core PP2Ac-A heterodimer (approximately 38- and approximately 65-kDa subunits, respectively) complexed with a putative, approximately 74-kDa B-type regulatory/targeting subunit. This heterotrimer lacks any strict substrate specificity in that it dephosphorylates C4 PEPC, mammalian phosphorylase a, and casein in vitro. This activity is independent of free Me2+, insensitive to levamisole and the Inhibitor-2 protein that targets PP1, activated by several polycations such as protamine and poly-L-lysine, and highly sensitive to inhibition by microcystin-LR and okadaic acid (IC50 approximately 30 pM), all of which are diagnostic features of yeast and mammalian PP2As. In addition, this C4-leaf PP2A holoenzyme (i) is inhibited in vitro by physiological concentrations of certain C4 PEPC-related metabolites (L-malate, PEP, glucose 6-phosphate, but not the activator glycine) when either 32P-labeled maize PEPC or rabbit muscle phosphorylase a is used as substrate, suggesting a direct effect on this Ser/Thr phosphatase; and (ii) displays, at best, only modest light/dark effects in vivo on its apparent molecular mass, component core subunits and activity against C4 PEPC, in marked contrast to the opposing activity of PEPC-kinase in C4 and Crassulacean acid metabolism leaves. This report represents one of the few studies of a heteromeric PP2A holoenzyme from photosynthetic tissue that dephosphorylates a known target enzyme in plants, such as PEPC, sucrose-phosphate synthase or nitrate reductase.     

The importance of the strictly conserved, C-terminal glycine residue in phosphoenolpyruvate carboxylase for overall catalysis: mutagenesis and truncation of GLY-961 in the sorghum C4 leaf isoform

Phosphoenolpyruvate carboxylase (PEPC) is a "multifaceted," allosteric enzyme involved in C4 acid metabolism in green plants/microalgae and prokaryotes. Before the elucidation of the three-dimensional structures of maize C4 leaf and Escherichia coli PEPC, our truncation analysis of the sorghum C4 homologue revealed important roles for the enzyme's C-terminal alpha-helix and its appended QNTG961 tetrapeptide in polypeptide stability and overall catalysis, respectively. Collectively, these functional and structural observations implicate the importance of the PEPC C-terminal tetrapeptide for both catalysis and negative allosteric regulation. We have now more finely dissected this element of PEPC structure-function by modification of the absolutely conserved C-terminal glycine of the sorghum C4 isoform by site-specific mutagenesis (G961(A/V/D)) and truncation (DeltaC1/C4). Although the C4 polypeptide failed to accumulate in a PEPC- strain (XH11) of E. coli transformed with the Asp mutant, the other variants were produced at wild-type levels. Although neither of these four mutants displayed an apparent destabilization of the purified PEPC homotetramer, all were compromised catalytically in vivo and in vitro. Functional complementation of XH11 cells under selective growth conditions was restricted progressively by the Ala, DeltaC1 and Val, and DeltaC4 modifications. Likewise, steady-state kinetic analysis of the purified mutant enzymes revealed corresponding negative trends in kcat and kcat/K0.5 (phosphoenolpyruvate) but not in K0.5 or the Hill coefficient. Homology modeling of these sorghum C-terminal variants against the structure of the closely related maize C4 isoform predicted perturbations in active-site molecular cavities and/or ion-pairing with essential, invariant Arg-638. These collective observations reveal that even a modest, neutral alteration of the PEPC C-terminal hydrogen atom side chain is detrimental to enzyme function.     

Comparative analysis of pyruvate kinases from the hyperthermophilic archaea Archaeoglobus fulgidus,Aeropyrum pernix,and Pyrobaculum aerophilum and the hyperthermophilic bacterium Thermotoga maritima: unusual regulatory properties in hyperthermophilic archaea

Pyruvate kinases (PK, EC 2.7.1.40) from three hyperthermophilic archaea (Archaeoglobus fulgidus strain 7324, Aeropyrum pernix, and Pyrobaculum aerophilum) and from the hyperthermophilic bacterium Thermotoga maritima were compared with respect to their thermophilic, kinetic, and regulatory properties. PKs from the archaea are 200-kDa homotetramers composed of 50-kDa subunits. The enzymes required divalent cations, Mg2+ and Mn2+ being most effective, but were independent of K+. Temperature optima for activity were 85 degrees C (A. fulgidus) and above 98 degrees C (A. pernix and P. aerophilum). The PKs were highly thermostable up to 110 degrees C (A. pernix) and showed melting temperatures for thermal unfolding at 93 degrees C (A. fulgidus) or above 98 degrees C (A. pernix and P. aerophilum). All archaeal PKs exhibited sigmoidal saturation kinetics with phosphoenolpyruvate (PEP) and ADP indicating positive homotropic cooperative response with both substrates. Classic heterotropic allosteric regulators of PKs from eukarya and bacteria, e.g. fructose 1,6-bisphosphate or AMP, did not affect PK activity of hyperthermophilic archaea, suggesting the absence of heterotropic allosteric regulation. PK from the bacterium T. maritima is also a homotetramer of 50-kDa subunits. The enzyme was independent of K+ ions, had a temperature optimum of 80 degrees C, was highly thermostable up to 90 degrees C, and had a melting temperature above 98 degrees C. The enzyme showed cooperative response to PEP and ADP. In contrast to its archaeal counterparts, the T. maritima enzyme exhibited the classic allosteric response to the activator AMP and to the inhibitor ATP. Sequences of hyperthermophilic PKs showed significant similarity to characterized PKs from bacteria and eukarya. Phylogenetic analysis of PK sequences of all three domains indicates a distinct archaeal cluster that includes the PK from the hyperthermophilic bacterium T. maritima.     

In vitro phosphorylation of phosphoenolpyruvate carboxylase from the green alga Selenastrum minutum

Previously, we described two distinct classes of phosphoenolpyruvate carboxylase (PEPC) isoforms in the green alga Selenastrum minutum. Class 1 PEPC (PEPC1) is a homotetramer composed of 102 kDa subunits (p102), whereas Class 2 PEPCs exist as three large protein complexes (PEPC2-PEPC4) containing varying proportions of structurally dissimilar p102 and 130 kDa (p130) PEPC catalytic subunits. In the current study, a p102 calcium-independent protein kinase was shown to co-purify with PEPC1, but not PEPC2. However, the p130 subunit of PEPC2 was phosphorylated in vitro during its incubation in the presence of [gamma-(32)P]ATP and a clarified algal extract. Treatment of purified PEPC2 with protein phosphatase 2A(2) increased its apparent M(r) as judged by Superose 6 gel filtration chromatography. The presence of the protein phosphatase inhibitors NaF and microcystin-LR throughout PEPC purification significantly influenced the activity and structural organization of Class 2, but not Class 1, PEPC isoforms. The results are consistent with the notion that under the culture conditions employed: (i) Class 1 and Class 2 PEPC isoforms exist in vivo mainly in their dephosphorylated and phosphorylated forms, respectively, and (ii) phosphorylation of Class 2 PEPCs leads to a significant reduction in their activity and native M(r). We propose that protein kinase-mediated phosphorylation is involved in the control and structural organization of green algal PEPC.     

Preferential Perinuclear Localization of Poly(ADP-ribose) Glycohydrolase

The transient nature of poly(ADP-ribosyl)ation, a posttranslational modification of nuclear proteins, is achieved by the enzyme poly(ADP-ribose) glycohydrolase (PARG) which hydrolyzes the poly(ADP-ribose) polymer into free ADP-ribose residues. To investigate the molecular size and localization of PARG, we developed a specific polyclonal antibody directed against the bovine PARG carboxy-terminal region. We found that PARG purified from bovine thymus was recognized as a 59-kDa protein, while Western blot analysis of total cell extracts revealed the presence of a unique 110-kDa protein. This 110-kDa PARG was mostly found in postnuclear extracts, whereas it was barely detectable in the nuclear fractions of COS7 cells. Further analysis by immunofluorescence revealed a cytoplasmic perinuclear distribution of PARG in COS7 cells overexpressing the bovine PARG cDNA. These results provide direct evidence that PARG is primarily a cytoplasmic enzyme and suggest that a very low amount of intranuclear PARG is required for poly(ADP-ribose) turnover.     

Insights into subunit interactions in the heterotetrameric structure of potato ADP-glucose pyrophosphorylase

ADP-glucose pyrophosphorylase, a key allosteric enzyme involved in higher plant starch biosynthesis, is composed of pairs of large (LS) and small subunits (SS). Current evidence indicates that the two subunit types play distinct roles in enzyme function. The LS is involved in mainly allosteric regulation through its interaction with the catalytic SS. Recently the crystal structure of the SS homotetramer has been solved, but no crystal structure of the native heterotetrameric enzyme is currently available. In this study, we first modeled the three-dimensional structure of the LS to construct the heterotetrameric enzyme. Because the enzyme has a 2-fold symmetry, six different dimeric (either up-down or side-by-side) interactions were possible. Molecular dynamics simulations were carried out for each of these possible dimers. Trajectories obtained from molecular dynamics simulations of each dimer were then analyzed by the molecular mechanics/Poisson-Boltzmann surface area method to identify the most favorable dimers, one for up-down and the other for side-by-side. Computational results combined with site directed mutagenesis and yeast two hybrid experiments suggested that the most favorable heterotetramer is formed by LS-SS (side-by-side), and LS-SS (up-down). We further determined the order of assembly during the heterotetrameric structure formation. First, side-by-side LS-SS dimers form followed by the up-down tetramerization based on the relative binding free energies.