Biochemical Characterization of the C4-Dicarboxylate Transporter DctA from Bacillus subtilis

ABSTRACT Bacterial secondary transporters of the DctA family mediate ion-coupled uptake of C4-dicarboxylates. Here, we have expressed the DctA homologue from Bacillus subtilis in the Gram-positive bacterium Lactococcus lactis. Transport of dicarboxylates in vitro in isolated membrane vesicles was assayed. We determined the substrate specificity, the type of cotransported ions, the electrogenic nature of transport, and the pH and temperature dependence patterns. DctA was found to catalyze proton-coupled symport of the four C4-dicarboxylates from the Krebs cycle (succinate, fumurate, malate, and oxaloacetate) but not of other mono- and dicarboxylates. Because (i) succinate-proton symport was electrogenic (stimulated by an internal negative membrane potential) and (ii) the divalent anionic form of succinate was recognized by DctA, at least three protons must be cotransported with succinate. The results were interpreted in the light of the crystal structure of the homologous aspartate transporter GltPh from Pyrococcus horikoshii.

The Endocytic Adaptor Protein ARH Associates with Motor and Centrosomal Proteins and Is Involved in Centrosome Assembly and Cytokinesis

Numerous proteins involved in endocytosis at the plasma membrane have been shown to be present at novel intracellular locations and to have previously unrecognized functions. ARH (autosomal recessive hypercholesterolemia) is an endocytic clathrin-associated adaptor protein that sorts members of the LDL receptor superfamily (LDLR, megalin, LRP). We report here that ARH also associates with centrosomes in several cell types. ARH interacts with centrosomal (γ-tubulin and GPC2 and GPC3) and motor (dynein heavy and intermediate chains) proteins. ARH cofractionates with γ-tubulin on isolated centrosomes, and γ-tubulin and ARH interact on isolated membrane vesicles. During mitosis, ARH sequentially localizes to the nuclear membrane, kinetochores, spindle poles and the midbody. Arh−/− embryonic fibroblasts (MEFs) show smaller or absent centrosomes suggesting ARH plays a role in centrosome assembly. Rat-1 fibroblasts depleted of ARH by siRNA and Arh−/− MEFs exhibit a slower rate of growth and prolonged cytokinesis. Taken together the data suggest that the defects in centrosome assembly in ARH depleted cells may give rise to cell cycle and mitotic/cytokinesis defects. We propose that ARH participates in centrosomal and mitotic dynamics by interacting with centrosomal proteins. Whether the centrosomal and mitotic functions of ARH are related to its endocytic role remains to be established.

Peroxynitrite and nitric oxide differ in their effects on pig coronary artery smooth muscle

Peroxynitrite generated in arteries from superoxide and nitric oxide (NO) may damage their function. Here, we compare the effects of peroxynitrite and peroxynitrite/NO-generating agents SIN-1 (3-morpholinosydnonimine hydrochloride), SNAP (S-nitroso- N-acetyl-penicillamine), SNP (sodium nitroprusside), and NONOate (spermine NONOate) on pig coronary artery. Deendothelialized artery rings were pretreated with these agents and then washed before examining their contractility. Pretreatment with all agents (200 μM) results in a decrease in the force of contraction in response to the sarco(endo)plasmic Ca2+ (SERCA) pump inhibitor cyclopiazonic acid (CPA): SNAP > NONOate ≥ peroxynitrite ≥ SIN-1 > SNP. Pretreatment with SNAP, NONOate, or SIN-1 also inhibits the force of contraction produced with 30 mM KCl, with SNAP being the most potent. Including catalase plus superoxide dismutase (SOD) during the preincubation has no effect. Including an NO scavenger [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide] or a guanylate cyclase inhibitor (1 H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) partially protects against SNAP. Pretreatment of cultured cells with peroxynitrite, but not with SNAP, inhibits the Ca2+transients produced in response to CPA. Pretreating isolated membrane vesicles with peroxynitrite inhibits the Ca2+ uptake due to the SERCA pump, with all the other agents being less effective. Thus peroxynitrite and NO both inhibit the CPA-induced contractions in deendothelialized artery rings, peroxynitrite by damage to the SERCA pump and NO possibly by a step downstream from the increase in cytosolic Ca2+.

Multidrug resistance protein Mrp2 mediates ATP-dependent transport of classic renal organic anion p-aminohippurate

p-Aminohippurate (PAH) is widely used as a model substrate to characterize organic anion transport in kidney proximal tubules. The carrier responsible for uptake of PAH across the basolateral membrane has been cloned and well characterized, whereas transporters mediating PAH excretion across the brush-border (apical) membrane are yet unknown. In this study we investigated whether PAH is a substrate for the apical multidrug resistance protein 2 (Mrp2). Overexpression of recombinant rabbit Mrp2 in Sf9 cells significantly increased ATP-dependent [14C]PAH uptake into isolated membrane vesicles compared with endogenous ATP-dependent uptake. The Michaelis-Menten constant and maximal velocity for Mrp2-mediated ATP-dependent [14C]PAH transport were 1.9 ± 0.8 mM and 187 ± 29 pmol · mg−1 · min−1, respectively. On the basis of the inhibitory profile, the endogenous ATP-dependent PAH transporter does not appear to be an ortholog of Mrp2. Together, our results show that Mrp2 is a low-affinity ATP-dependent PAH transporter, indicating that Mrp2 might contribute to urinary PAH excretion.

Glucocorticoid regulation of splanchnic glutamine, alanine, glutamate, ammonia, and glutathione fluxes

Interorgan glutamine and associated metabolite fluxes were measured across the gut and liver to delineate splanchnic bed fluxes secondary to enhanced arterial loads mobilized in the periphery by glucocorticoid. Experiments were performed on adrenalectomized rats since adrenalectomy doubled the hepatic glucocorticoid receptor population compared with intact animals. Under these conditions, triamcinolone supplement (40 micrograms.day-1.100 g body wt-1) enhanced the combined net glutamine uptake by gut and liver eightfold, whereas combined gut and liver unidirectional breakdown and synthesis fluxes both increased (3.4- and 7.4-fold, respectively). Triamcinolone supplement also altered the pattern of metabolite released; gut released predominantly ammonium and some alanine, whereas the liver removed more alanine along with glutamine and released more urea, glutamate, and glutathione. Mechanistically, enhanced cellular glutamine uptake could be attributed to a three- to fourfold acceleration of glutamine transport associated with a rise in intracellular glutamine content. However, uptake by isolated membrane vesicles revealed only a small (27%) increase in System N activity, whereas extraction and reconstitution of the transporter into proteoliposomes failed to demonstrate increased transporter activity. Similarly, activity of phosphate-dependent glutaminase and glutamate dehydrogenase increased in crude homogenates (2-fold), but the former disappears in completely disrupted preparations. Furthermore, whereas messenger RNA and assayable enzymic activity for glutamate dehydrogenase clearly increased with glucocorticoid, glutaminase message was less significantly increased. Thus glucocorticoid appears directly capable of accelerating hepatic glutamine extraction primarily by modulating transporter activity that is closely coupled to glutamine utilization.