Uptake Of L-malate Research Articles

C4-dicarboxylate metabolons: interaction of C4-dicarboxylate transporters of Escherichia coli with cytosolic enzymes.

Metabolons represent the structural organization of proteins for metabolic or regulatory pathways. Here, the interaction of fumarase FumB, aspartase AspA, and L-tartrate dehydratase TtdAB with the C4-dicarboxylate (C4-DC) transporters DcuA, DcuB, DcuC, and the L-tartrate transporter TtdT of Escherichia coli was tested by a bacterial two-hybrid (BACTH) assay in situ, or by co-chromatography using mSPINE (membrane Streptavidin protein interaction experiment). From the general C4-DC transporters, DcuB interacted with FumB and AspA, DcuA with AspA, whereas DcuC interacted with neither FumB nor AspA. Moreover, TtdT did not interact with TtdAB. The fumB-dcuB, the dcuA-aspA, and the ttdAB-ttdT genes encoding the respective proteins colocalize on the genome and each pair of genes forms cotranscripts, whereas the dcuC gene lies alone. The data suggest the formation of DcuB/FumB and DcuB/AspA metabolons for the uptake of L-malate, or L-aspartate, and their conversion to fumarate for fumarate respiration and excretion of the product succinate. The DcuA/AspA metabolon catalyzes uptake and conversion of L-aspartate to fumarate coupled to succinate excretion. The DcuA/AspA metabolon provides ammonia at the same time for nitrogen assimilation (ammonia shuttle). On the other hand, TtdT and TtdAB are not organized in a metabolon. Reasons for the formation (DcuA/AspA, DcuB/FumB, and DcuB/AspA) or nonformation (DcuC, TtdT, and TtdAB) of metabolons are discussed based on their metabolic roles.

High-affinity l-malate transporter DcuE of Actinobacillus succinogenes catalyses reversible exchange of C4-dicarboxylates.

Actinobacillus succinogenes is a natural succinate producer, which is the result of fumarate respiration. Succinate production from anaerobic growth with C4 -dicarboxylates requires transporters catalysing uptake and efflux of C4 -dicarboxylates. Transporter Asuc_1999 (DcuE) found in A. succinogenes belongs to the Dcu family and was considered the main transporter for fumarate respiration. However, deletion of dcuE affected l-malate uptake of A. succinogenes rather than fumarate uptake. DcuE complemented anaerobic growth of Escherichia coli on l-malate or fumarate; thus, the transporter was characterized in E. coli heterologously. Time-dependent uptake and competitive inhibition assays demonstrated that l-malate is the most preferred substrate for uptake by DcuE. The Vmax of DcuE for l-malate was 20.04 μmol/gDW·min with Km of 57 μM. The Vmax for l-malate was comparable to that for fumarate, whereas the Km for l-malate was 8 times lower than that for fumarate. The catalytic efficiency of DcuE for l-malate was 7.3-fold higher than that for fumarate, showing high efficiency and high affinity for l-malate. Furthermore, DcuE catalysed the reversible exchange of three C4 -dicarboxylates - l-malate, fumarate and succinate - but the preferred substrate for uptake was l-malate. Under physiological conditions, the C4 -dicarboxylates were reduced to succinate. Therefore, DcuE is proposed as the l-malate/succinate antiporter in A. succinogenes.

Amonafide L-Malate Bypasses Multidrug Resistance Proteins in Secondary Acute Myeloid Leukemia.

Amonafide L-malate, a DNA intercalating agent and ATP-independent topoisomerase 2 inhibitor showed promising clinical activity in combination with cytarabine in a Phase 2 clinical trial in secondary acute myeloid leukemia (S-AML), with a complete remission (CR) rate of 45% (Erba, et al., JCO 25:373s, 2007), and is currently in a Phase 3 clinical trial. We hypothesized that the activity of amonafide L-malate in S-AML may be attributable in part to its being a poor substrate for the multidrug resistance (MDR)-associated drug efflux proteins expressed in AML cells, including P-glycoprotein (Pgp; MDR1; ABCB1), multidrug resistance protein-1 (MRP-1;ABCC1) and breast cancer resistance protein (BCRP;ABCG2). Amonafide has been previously shown not to be a substrate for Pgp in cell lines overexpressing this protein (Chau, et al., Leuk Res, in press). Here we studied transport and cytotoxicity of amonafide L-malate and its active metabolite N-acetyl amonafide in cell lines overexpressing Pgp, MRP-1 and BCRP and in pretreatment blasts from patients with S-AML. Cellular drug transport was studied by flow cytometry and cytotoxicity was studied in 96-well microcultures in the absence and presence of the MDR modulators PSC-833 (Pgp), probenecid (MRP-1) and fumitremorgin C (FTC), and were compared with those of classical topoisomerase 2 inhibitors including doxorubicin, daunorubicin, idarubicin, mitoxantrone and etoposide in leukemia and myeloma cell lines overexpressing Pgp, MRP-1 and BCRP and in pretreatment marrow samples from 22 S-AML patients characterized with regard to expression and function of these proteins. S-AML patients included 9 with therapy-related AML and 13 with antecedent myelodysplastic syndromes. Pgp, MRP-1 and BCRP expression in pretreatment blasts was measured by flow cytometry with the MRK16, MRPm6 and BXP21 antibodies, and function by uptake of the fluorescent substrates DiOC2(3), rhodamine-123 and pheophorbide A with modulation by PSC-833, probenecid and FTC, respectively. Uptake, efflux and cytotoxicity of amonafide L-malate did not differ in resistant cell lines overexpressing Pgp, MRP-1 and BCRP, compared to parental cells, and did not differ in the presence and absence of MDR modulators, in contrast to all of the other topoisomerase 2 inhibitors studied. Cell lines overexpressing Pgp and MRP-1 did not display transport of, or resistance to, the metabolite, acetyl amonafide, but acetyl amonafide was a substrate for BCRP, with 8-fold resistance in cells with BCRP expression, and 8-fold sensitization by FTC. Pgp, MRP-1 and BCRP expression and/or function was observed in 18, 7 and 17 of 22 secondary AML samples, respectively. Cyclosporin A, which inhibits substrate drug efflux by Pgp, MRP-1 and BCRP, increased uptake of daunorubicin, idarubicin and amonafide L-malate by mean values of 19.7%, 7% and −2.5%, respectively, and increased uptake by ≥ 10% in 16, 12 and 5 patient samples. In conclusion, in relation to other topoisomerase 2 inhibitors used to treat AML, including daunorubicin, idarubicin, mitoxantrone, and etoposide, amonafide L-malate is a poor substrate for the MDR proteins expressed in AML cells in general, and S-AML cells in particular. The encouraging complete remission rates seen with amonafide L-malate treatment in clinical trials in S-AML may thus be attributed at least in part to its potential to overcome MDR in S-AML cells.

Differential uptake of fumarate by Candida utilis and Schizosaccharomyces pombe.

The dicarboxylic acid fumarate is an important intermediate in cellular processes and also serves as a precursor for the commercial production of fine chemicals such as L-malate. Yeast species differ remarkably in their ability to degrade extracellular dicarboxylic acids and to utilise them as their only source of carbon. In this study we have shown that the yeast Candida utilis effectively degraded extracellular fumarate and L-malate, but glucose or other assimilable carbon sources repressed the transport and degradation of these dicarboxylic acids. The transport of both dicarboxylic acids was shown to be strongly inducible by either fumarate or L-malate while kinetic studies suggest that the two dicarboxylic acids are transported by the same transporter protein. In contrast, Schizosaccharomyces pombe effectively degraded extracellular L-malate, but not fumarate, in the presence of glucose or other assimilable carbon sources. The Sch. pombe malate transporter was unable to transport fumarate, although fumarate inhibited the uptake of L-malate.

The mae1 gene of Schizosaccharomyces pombe encodes a permease for malate and other C4 dicarboxylic acids.

The mae1 gene of the yeast Schizosaccharomyces pombe was identified on the basis of its ability to complement a mutant defective in the transport of malic acid. Analysis of the DNA sequence revealed an open reading frame of 1314 base pairs, encoding a polypeptide of 438 amino acids with a predicted molecular weight of 49 kDa. A hydropathy profile of the predicted amino acid sequence revealed a protein with ten membrane-spanning or associated domains and hydrophilic N- and C- termini. The predicted secondary structure of the protein in similar to models proposed for other integral membrane proteins from both prokaryotes and eukaryotes. The S. pombe mae1 gene encodes a single mRNA of 1.5 kb. The mea1 gene is expressed constitutively and is not subject to catabolite repression as was previously reported for the malate permease systems of Candida utilis and Hansenula anomala. The mae1 gene was mapped 2842 bp 5' to the MFml gene on chromosome I. Transport assays revealed that the mae1 gene encodes a permease involved in the uptake of L-malate, succinate and malonic acid.

Uniport of monoanionic L-malate in membrane vesicles from Leuconostoc oenos.

L-malate transport was studied in membrane vesicles from Leuconostoc oenos MLE(-) (mutant lacking malolactic enzyme) which were fused with liposomes containing beef heart cytochrome c oxidase as a proton-motive-force-generating system. In these hybrid membranes, accumulation of L-malate was observed in response to a pH gradient (delta pH), with the inside alkaline, but was strongly inhibited by a membrane potential (delta psi) of normal polarity (inside negative). Imposition of a delta psi, with the inside positive, by means of valinomycin-mediated potassium influx, resulted in a rapid accumulation of L-malate, indicating that L-malate was taken up in an anionic form. The results are consistent with a uniport mechanism facilitating the uptake of monoanionic L-malate, the dominant species at the low pH of the experiments. Kinetic analysis of delta pH-driven L-malate uptake in the pH range 3.0-5.8, yielded apparent affinity constants that varied less than twofold when calculated on the basis of the concentrations of monoanionic L-malate, whereas the values differed 2-3 orders of magnitude for the other species. At L-malate concentrations above 1 mM, a non-saturable transport component became apparent which may reflect passive influx of L-malic acid. Substrate specificity studies indicated that citrate and L-malate (and possibly D-lactate and L-lactate) compete for a single general carboxylate transport system. The carboxylate transport system catalysed homologous L-malate and heterologous L-malate/citrate exchange with rates similar to the rate of L-malate efflux. Since metabolic energy is conserved during malolactic fermentation in L. oenos, the underlying mechanism most likely involves electrogenic monoanionic L-malate uptake, in combination with H+ consumption in the cytoplasm, followed by diffusion outwards of lactic acid plus carbon dioxide.

Obtaining functional membrane vesicles from Leuconostoc oenos to study l-malate transport mechanisms

Membrane vesicles from the malolactic bacterium Leuconostoc oenos were obtained by a modified version of the procedure of Kaback [Methods Enzymol 22:99–120 (1971)]. Protoplasts were produced at frequencies greater than 95% by a method entailing mutanolysis digestion and osmotic shock. Glycerol or polyethyleneglycol 600 was required as an osmotic stabilizer while the use of sucrose prevented closed vesicle formation during osmotic shock. The membrane vesicles retained their functional properties and accumulated l-malic acid in response to an ATPase-induced proton gradient across the membrane of ATP-loaded vesicles. l-Malate uptake was strongly inhibited by dicyclohexylcarbodiimide, a specific inhibitor of membrane-bound ATPase. These data support the possibility of a ΔpH-dependent transport of l-malate. Vesicles not loaded with ATP were slightly permeable to malic acid with an initial uptake rate (0.5 nmol·μl−1·s−1) similar to the diffusion rate obtained previously in a L. oenos malate-transport-deficient strain. These results confirm two simultaneous uptake mechanisms in L. oenos, a permease-mediated transport and a passive diffusion for the anionic and the undissociated forms of l-malic acid respectively.

Malolactic fermentation: electrogenic malate uptake and malate/lactate antiport generate metabolic energy

The mechanism of metabolic energy production by malolactic fermentation in Lactococcus lactis has been investigated. In the presence of L-malate, a proton motive force composed of a membrane potential and pH gradient is generated which has about the same magnitude as the proton motive force generated by the metabolism of a glycolytic substrate. Malolactic fermentation results in the synthesis of ATP which is inhibited by the ionophore nigericin and the F0F1-ATPase inhibitor N,N-dicyclohexylcarbodiimide. Since substrate-level phosphorylation does not occur during malolactic fermentation, the generation of metabolic energy must originate from the uptake of L-malate and/or excretion of L-lactate. The initiation of malolactic fermentation is stimulated by the presence of L-lactate intracellularly, suggesting that L-malate is exchanged for L-lactate. Direct evidence for heterologous L-malate/L-lactate (and homologous L-malate/L-malate) antiport has been obtained with membrane vesicles of an L. lactis mutant deficient in malolactic enzyme. In membrane vesicles fused with liposomes, L-malate efflux and L-malate/L-lactate antiport are stimulated by a membrane potential (inside negative), indicating that net negative charge is moved to the outside in the efflux and antiport reaction. In membrane vesicles fused with liposomes in which cytochrome c oxidase was incorporated as a proton motive force-generating mechanism, transport of L-malate can be driven by a pH gradient alone, i.e., in the absence of L-lactate as countersubstrate. A membrane potential (inside negative) inhibits uptake of L-malate, indicating that L-malate is transported an an electronegative monoanionic species (or dianionic species together with a proton). The experiments described suggest that the generation of metabolic energy during malolactic fermentation arises from electrogenic malate/lactate antiport and electrogenic malate uptake (in combination with outward diffusion of lactic acid), together with proton consumption as result of decarboxylation of L-malate. The net energy gain would be equivalent to one proton translocated form the inside to the outside per L-malate metabolized.

Transport of pyruvate by luminal membrane vesicles from pars convoluta and pars recta of rabbit proximal tubule

The characteristics of renal transport of pyruvate by luminal membrane vesicles from pars convoluta and pars recta of rabbit proximal tubule were studied. It was found that the uptake of pyruvate in these vesicle preparations occurred by means of multiple transport systems. An electrogenic and Na +-requiring system confined to pars convoluta, exists for transport of pyruvate with an intermediate affinity, K A = 0.71 ± 0.08 mM . In vesicles from pars recta, the uptake of pyruvate was mediated by a dual transport system with a high ( K A1 = 0.30 ± 0.05 mM ) and low affinity ( K A2 = 5.75 ± 0.82 mM ). The relation of these three pyruvate transport systems to the transport of other monocarboxylates and dicarboxylates was determined by examination of the inhibitory effect of l-lactate, β-hydroxybutyrate and l-malate on the renal uptake of pyruvate. It was found that l-malate efficiently reduced the uptake of pyruvate by vesicles from pars convoluta, while addition of monocarboxylates ( l-lactate, β-hydroxybutyrate) has no effect on the transport of pyruvate in this region of proximal tubule. Furthermore, it was observed that Na +-dependent uptake of l-malate was specifically inhibited to about the same extent by 1 mM pyruvate. These findings strongly suggest that pyruvate and l-malate are taken up the same transport system in vesicles from pars convoluta, which is different from that of l-lactate and β-hydroxybutyrate. Experiments designed to investigate the substrate specificity for the high and low affinity system for pyruvate in pars recta, revealed that monocarboxylates ( l-lactate, β-hydroxybutyrate) substantially inhibit pyruvate transport at the low, but not the high substrate concentration, indicating that all monocarboxylates compete for the same high-affinity transport system. By contrast, l-malate at low concentrations preferentially inhibited the low-affinity system for pyruvate. This observation led us to suggest that l-malate and pyruvate share a common transport system in pars recta with low affinity for pyruvate and high affinity for l-malate.

Mechanism of L-malate transport in rat renal basolateral membrane vesicles.

The transport of L-malate was evaluated in basolateral and brush border membrane vesicles isolated from the rat renal cortex. In both types of membrane vesicles, an inwardly directed sodium gradient stimulated transport and caused an overshoot of the equilibrium concentration. In basolateral membrane vesicles, the presence of sodium, but without a sodium gradient, stimulated the uptake of L-malate. Sodium gradient-stimulated L-malate uptake was stimulated and inhibited by maneuvers designed to render the inside of the basolateral membrane vesicles more electronegative or electropositive, respectively. The tricarboxylic acid cycle intermediates citrate, alpha-ketoglutarate, succinate, fumarate, L-malate, and oxaloacetate and the monocarboxylic acid pyruvate inhibited the sodium gradient-stimulated uptake of L-[14C]malate in basolateral membrane vesicles. Maleate, gluconate, formate, acetate, L-lactate, and beta-hydroxybutyrate did not inhibit uptake. These results indicate that basolateral membrane vesicles from the rat renal cortex contain a sodium-L-malate cotransport system that may be shared by the other tricarboxylic acid cycle intermediates and by pyruvate. Such a transport system, together with a similar sodium-tricarboxylic acid cycle intermediate cotransport system located in the brush border membrane, would ensure high concentrations of tricarboxylic acid cycle intermediates within the cells of the proximal tubule.

A Comparative Study of Citrate Efflux from Mitochondria of Oleaginous and Non‐oleaginous Yeasts

Citrate efflux from mitochondria of 10 different yeasts was investigated. Isolated mitochondria of all the yeasts show the possession of a carrier system for dicarboxylic and tricarboxylic anions, phosphate and pyruvate. The tricarboxylate carrier is also specific for l‐malate in all 10 yeasts. In citrate‐loaded mitochondria, citrate efflux and l‐malate uptake are directly proportional to the concentration of extramitochondrial l‐malate until the translocator becomes saturated (> 8 mM l‐malate in Candida curvata D). Rates of citrate efflux in the presence of l‐malate are linear for a period of 2 min and are approximately 2.5‐times greater in oleaginous than in non‐oleaginous yeasts. The malate‐citrate translocator has a significantly lower Km for l‐malate in oleaginous yeasts (∼ 4.0 mM) than in non‐oleaginous yeasts (∼ 8.4 mM). Both pyruvate and phosphate appear to stimulate the malate‐citrate exchange in mitochondria of C. curvata D. Oleginous yeast mitochondria contain 3—4‐times higher intramitochondrial citrate levels than non‐oleaginous yeast. In all these yeasts the enzymes for synthesis and metabolism of citrate were exclusively mitochondrial, the only exception being the possession of a cytosolic ATP; citrate lyase by oleaginous yeasts. In metabolically active mitochondria, once the malate‐citrate carrie is saturated with l‐malate, efflux of citrate is directly proportional to the concentration of extramitochondrial pyruvate (as acetyl donor). The rates of efflux of newly synthesised citrate from metabolically active mitochondria, in the presence of saturating l‐malate and pyruvate, are 6–8‐times greater in oleaginous yeasts and the citric‐acid‐producing yeast. The inhibition of aconitase by fluorocitrate produced a ninefold increase in citrate efflux from non‐oleaginous yeast mitochondria but only a slight increase in oleaginous yeasts. It is concluded that in fully functional yeast mitochondria, citrate efflux is limited by the amount of intra‐mitochondrial citrate made available to the citrate translocator for exchange.

An efficient method for the isolation and separation of basolateral-membrane and luminal-membrane vesicles from rabbit kidney cortex.

A procedure for isolation and separation of purified luminal-membrane and basolateral-membrane vesicles from adult and newborn rabbit renal cortex by using Ca2+/Mg2+ precipitation, differential centrifugation and a self-orienting Percoll-gradient centrifugation is described. The purity of the membrane-vesicle suspensions was examined by electron microscopy and by measuring the activity of several marker enzymes. The activity of Na+ + K+-stimulated ATPase in the fraction mainly containing adult rabbit basolateral-membrane vesicles was enriched 16-fold, and the activity of alkaline phosphatase in the fraction mainly containing luminal-membrane vesicles was increased 13-fold, compared with the homogenate. Similar results were obtained with kidneys from newborn rabbits. Uptake studies, with a rapid filtration technique and the spectrophotometric method described in an accompanying paper [Kragh-Hansen, Jørgensen & Sheikh (1982) Biochem. J. 208, 359-368], showed that both adult and newborn rabbit luminal-membrane vesicles, in contrast with the basolateral-membrane preparations, possess an Na+-dependent electrogenic transport system for L-proline. Adult rabbit luminal-membrane vesicles take up citrate and L-malate by Na+-dependent electrogenic processes, whereas adult rabbit basolateral membrane vesicles do not exhibit electrogenic uptake of citrate. By contrast, these vesicles show Na+-dependent electrogenic uptake of L-malate.

Efficiency of light-driven metabolite transport in the photosynthetic bacterium Rhodospirillum rubrum.

An evaluation of the efficiency of the L-alanine and L-malate transport systems was undertaken with the photosynthetic bacterium Rhodospirillum rubrum grown on the amino acid whose uptake was measured. An all-glass apparatus was constructed for measuring transport activity under anaerobic conditions. L-Alanine transport activity decreased under conditions of Mg2+ depletion. When cells were allowed to become inactive by suspending them in the dark in Mg2+-free buffer, full activity could be restored with a few minutes by adding 20 mM Mg2+ and illuminating the cells. The transport activity was completely inhibited by carbonyl cyanide m-trifluoromethoxyphenylhydrazone and by ammonia. The quantum yield for the uptake of either L-alanine or L-malate was 0.015 molecules per photon. The results are discussed in relation to the expected efficiencies for metabolite transport and regulation by Mg2+.

Respiration-driven accumulation of C4 dicarboxylic acids by isolated membrane vesicles of Paracoccus denitrificans

Membrane vesicles derived by osmotic lysis of spheroplasts of Paracoccus denitrificans (ATCC 13543) grown aerobically in continuous culture under conditions of carbon limitation with succinate as sole carbon and energy source accumulate radioactivity in an uncoupler-sensitive respiration-dependent manner when incubated in the presence of [14C]succinate or L-[14C]malate. Membranes prepared from cells grown under conditions of sulphate limitation with succinate as the sole carbon source do not accumulate L-[14C]malate during ascorbate + N,N,N′,N′-tetramethyl-p-phenylene diamine (TMPD) oxidation. The apparent Km for succinate and L-malate uptake is 6.7 and 10 μM respectively with a Vmax for uptake of either substrate being 8.3 nmol/min per milligram of membrane protein. Intravesicular radioactivity was largely confined to C4 dicarboxylic acids, succinate, fumarate, and malate and was freely exchangeable with external unlabeiled C4 dicarboxylic acids but not aspartate. Both ubiquinol1 and ascorbate (+ TMPD) oxidation supported accumulative uptake of succinate or L-malate but NADH did not. Since energization of the dicarboxylate transport system is accomplished by the oxidation of ascorbate + TMPD in the presence of antimycin A, it is concluded that heterotrophically grown P. denitrificans containing cytochrome a3 possess a functional energy-coupling site 3 (terminal energy-transducing region of the respiratory chain) despite claims to the contrary.

The transport of dicarboxylates and some properties of fumarase in the muscle mitochondria of Ascaris suum

1. 1. Muscle mitochondria of the parasitic nematode Ascaris suum showed a swelling effect when suspended in iso-osmotic solutions of NH 4 L-malate, NH 4 succinate and NH 4 malonate. This process was stimulated by low concentrations of inorganic phosphate, but completely inhibited by 2- n-pentylmalonate and p-iodobenzylmalonate. Very little or no swelling was observed with NH 4 fumarate, even when catalytic amounts of phosphate were present. 2. 2. All dicarboxylates including fumarate elicited efflux of intramitochondrial malonate. This effect was found to be sensitive to transport inhibitors. 3. 3. By comparing the time course of malonate efflux caused by L-malate and fumarate it was found that the former anion was taken up very rapidly following hyperbolic kinetics, whereas the comparably slight effect of fumarate resulted in a sigmoidal curve. 4. 4. The experimental data indicate that the rate of malonate efflux caused by fumarate was actually due to an uptake of L-malate which has been shown to be rapidly formed by fumarase, externally to the membrane barrier in the mitochondrial incubations. 5. 5. It is concluded that fumarate appears to be not able to penetrate the mitochondrial permeability barrier. On the other hand, conclusive evidence has been obtained that the transport of L-malate, succinate and malonate in Ascaris muscle mitochondria is carrier mediated as is the case for mammalian mitochondria.

Evidence for Inducible, l-Malate Binding Proteins in the Membrane of Bacillus subtilis: IDENTIFICATION OF PRESUMPTIVE COMPONENTS OF THE C4-DICARBOXYLATE TRANSPORT SYSTEMS

Abstract l-Malate was found to protect inducible C4-dicarboxylate transport in Bacillus subtilis against the inhibitory effects of the irreversible sulfhydryl reagent N-ethylmaleimide. This effect served as the basis for specifically B. subtilis with N-ethylmaleimide according to a procedure originally employed by Fox and Kennedy (Fox, C.F. and Kennedy, E. P. (1965) Proc. Nat. Acad. Sci. U.S.A. 54, 891) for N-ethylmaleimide labeling the M-protein component of the lactose transport system of Escherichia coli. By means of this method, three proteins were detected in the membranes of l-malate-induced cells, but not of uninduced cells. These proteins were protected against reaction with N-ethylmaleimide by the presence of saturating amounts of l-malate. The electrophoretic mobilities of these proteins on polyacrylamide gels in the presence of sodium dodecyl sulfate suggested they had subunit weights of 62,000, 44,000, and 33,000. That they were quantitatively minor components of the membrane was evidenced by the finding that direct labeling with amino acids did not consistently detect the presence of these proteins in the membrane of induced cells. Studies with substrate analogues established a direct correlation between the ability of a compound to competitively inhibit l-malate uptake and the ability of that same compound to protect against N-ethylmaleimide inactivation of C4-dicarboxylate transport activity. Moreover, meso-tartrate, a powerful competitive inhibitor of l-malate uptake, was capable not only of protecting transport activity against N-ethylmaleimide inactivation but also of protecting the 62,000- and 44,000-dalton proteins against reaction with N-ethylmaleimide. The 33,000-dalton protein was not protected against reaction with N-ethylmaleimide by meso-tartrate. l(+)-Tartrate, a poor competitive inhibitor of l-malate uptake, protected neither transport activity nor the 62,000-, 44,000-, or 33,000-dalton proteins from N-ethylmaleimide. A possible role for these proteins in the transport of dicarboxylic acids into B. subtilis is discussed.

Biogenesis of mitochondria. The effects of physiological and genetic manipulation of Saccharomyces cerevisiae on the mitochondrial transport systems for tricarboxylate-cycle anions

1. Kinetic and equilibrium parameters for the uptake of l-malate, succinate, citrate and alpha-oxoglutarate by fully functional mitochondria of Saccharomyces cerevisiae were determined. 2. The uptake of l-malate and succinate is mediated by a common carrier, and two other distinct carriers mediate the uptake of citrate and alpha-oxoglutarate. 3. The properties of the carrier systems for l-malate, succinate and citrate closely resemble those of mammalian mitochondria, but the alpha-oxoglutarate carrier differs from the mammalian system in minor respects. 4. The composition of the yeast mitochondria was extensively manipulated by (a) anaerobiosis, (b) catabolite repression, (c) inhibition of mitochondrial protein synthesis and (d) elimination of mitochondrial DNA by mutation. 5. The carrier systems for l-malate, succinate, citrate and alpha-oxoglutarate are essentially similar in the five different types of mitochondria. 6. It is concluded that all the protein components of the carrier systems for l-malate, succinate, citrate and alpha-oxoglutarate are coded by nuclear genes and synthesized extramitochondrially by cell-sap ribosomes.

Transport of C 4-dicarboxylic acids in Neurospora crassa

1. 1. The transport of tricarboxylic acid cycle dicarboxylic acids by Neurospora crassa was induced 80-fold by growth on acetate whereas tricarboxylic acid enzymes were induced only 2-6-fold and glyoxylate cycle enzymes were induced 20-fold. 2. 2. From the kinetics of uptake and competitive inhibition it was concluded that a single system was responsible for the transport of succinate, fumarate, l-malate and possibly α-ketoglutarate as well as a number of structural analogues. The system appears to be less specific than the known bacterial systems. 3. 3. Succinate transport was strongly inhibited by various known inhibitors of energy metabolism — primarily by proton conductors. Transport was also severely inhibited by various sulfhydryl reagents. 4. 4. Succinate, fumarate and l-malate uptake were identically pH dependent and evidence is presented which implicates the monoionic species as the molecular form of the dicarboxylic acid transported.

Transport of Dicarboxylic Acids in Bacillus subtilis: INDUCIBLE UPTAKE OF l-MALATE

Abstract The uptake of several dicarboxylic acids by Bacillus subtilis has been surveyed. Gratuitous conditions for induction of l-malate uptake were achieved under which the addition of l-malate to the medium resulted in increased uptake of l-malate by the cells without significantly influencing the growth rate. The specificity of induction of l-malate uptake was investigated using other dicarboxylate anions of the tricarboxylic acid cycle. Fumarate induced l-malate uptake with about ⅓ the efficiency of l-malate. No induction of l-malate uptake was observed in a succinic dehydrogenase (EC 1.3.99.1) negative mutant grown in the presence of succinate, although this strain could be fully induced for l-malate uptake by the addition of l-malate to the growth medium. l-Malate uptake in a B. subtilis mutant lacking malic dehydrogenase (EC 1.1.1.37) activity appeared constitutive, probably owing to endogenous induction caused by the accumulation of l-malate inside the cells. These data suggested that l-malate was the true inducer of the l-malate transport system, but confirmation of this notion must come from a system in which l-malate is strictly nonmetabolizable. The l-malate transport system could be catabolite repressed by d-glucose. l-Malate uptake followed simple Michaelis-Menten kinetics; its apparent Km was 0.4 mm and Vmax approached 400 µmoles per min per g dry weight of cells. The system was shown to be highly specific by using competitive inhibitors, of which l(+)citramalate, mesotartrate,3-fluoro-dl-malate, and d(+)-malate were most effective. 2,4-Dinitrophenol, azide, and classical uncouplers of oxidative phosphorylation strongly inhibited l-malate uptake in B. subtilis. The l-malate transport system was different from the previously reported citrate uptake system: the two systems could be separately induced. Furthermore, mutants deficient in citrate uptake largely retained the ability to be induced for l-malate uptake. Fumarate uptake in B. subtilis was inducible to fairly high activities but seemed dependent on the continual removal of fumarate via the tricarboxylic acid cycle in order to exhibit maximal uptake rates. The Km for the process was 0.7 mm. Similarly, α-ketoglutarate was transported by an inducible system; the process had a high Km (6.7 mm). We were unable to demonstrate induction of a system for succinate uptake, and saturation kinetics were not observed over the 50-fold concentration range tested (0.1 to 5.0 mm). dl-Tartrate uptake could be strongly inhibited by the addition of l-malate or l(+)tartrate to the medium during growth.