The kinetics of citrate uptake by malate-loaded mitochondria were measured using the inhibitor stop method and analysed for possible carrier mechanisms. 1.The citrate exchange is found to follow a first order reaction with a constant k of 1.18 min-l and the corresponding rate of citrate uptake of 13.4 pmolxmin-l x g protein-l (at 0.5 mM citrate and 9 "C). The half time is 34 see.2. The temperature dependence of citrate exchange was measured in the range between 0 and 14 "C. An activation energy of 20.1 kcal and a Q,, of3.6 can be calculated from the Arrhenius 3. The concentration dependence of citrate exchange reveals hyperbolic saturation characteristics. The K , and V values for the rate of citrate uptake are 0.12 f 0.01 mM and 22.5 & 1.8 pmol citrate x mir-l x g protein-l respectively at 9 "C in 11 experiments. 4.The rate of citrate uptake has a pH optimum of about 7. The inhibition by higher pH is competitive with citrate. At pH lower than 7, V is decreased, indicating that the citrate transporting system is inactivated by acid pH.5. The rate of citrate exchange is inhibited in a competitive manner by cis-aconitate, threo-D,-isocitrate, 1,2,3-propanetncarboxylate, 1,2,3-benzenetricarboxylate, citrate and propylcitrate. Other tricarboxylates, however, such as trans-aconitate and 1,3,5-pentanetricarboxylate have no effect on citrate exchange, even when added in large excess.6. Succinate, malonate, oxaloacetate and oxomalonate inhibit the rate of citrate uptake. In contrast, glutarate, adipate, pimelate, 2-oxoglutarate, aspartate and glutamate do not. Maleate, but not the trans isomer fumarate, also inhibits citrate uptake. The inhibition of citrate uptake is great,er with malate than with succinate.7. The rate of citrate uptake is also inhibited by the nonpenetrant anions phenylsuccinate, butylmalonate, benzylmalonate and pentylmalonate, previously thought to be specsc inhibitors of the dicarboxylate carrier.8. The inhibition of the rate of citrate uptake by dicarboxylates and their analogues is found to be competitive. The affinity for dicarboxylates is lower than for tricarboxylates (Ki is 0.7 mM for malate).9. Phosphoenolpyruvate strongly inhibits the rate of citrate uptake, while Pi and pyruvate have only a slight effect. The inhibition by phosphoenolpyruvate is shown to be competitive (Ki is 0.11 mM). 10.It is concluded that the tricarboxylate carrier has a single binding site for tricarboxylates, plot.phosphoenolpyruvate and dicarboxylates. The implications of these findings are discussed.Several pieces of evidence indicate that the transport of tricarboxylates through the mitochondria1 membrane is mediated by a specific carrier, which catalyzes an exchange diffusion of a tricarboxylate for either a tricarboxylate or a dicarboxylate [1,2].To gain further insight into the catalytic mechanism and for a quantitative understanding of the physiological importance of the ticarboxylate carrier, direct kinetic studies are necessary.In this paper, quantitative values of the rate and related kinetic parameters of ...

Using a functional mitochondrial oxoglutarate carrier mutant devoid of Cys residues (C-less carrier), each amino acid residue in transmembrane domain IV and flanking hydrophilic loops (from T179 to S205) was replaced individually with Cys. The great majority of the 27 mutants exhibited significant oxoglutarate transport in reconstituted liposomes as compared to the activity of the C-less carrier. In contrast, Cys substitution for G183, R190, Q198, and Y202, in either C-less or wild-type carriers, yielded molecules with complete loss of oxoglutarate transport activity. G183 and R190 could be partially replaced only by Ala and Lys, respectively, whereas Q198 and Y202 were irreplaceable with respect to oxoglutarate transport. Of the single-Cys mutants tested, only T187C, A191C, V194C, and N195C were strongly inactivated by N-ethylmaleimide and by low concentrations of methanethiosulfonate derivatives. Oxoglutarate protects Cys residues at positions 187, 191, and 194 against reaction with N-ethylmaleimide. These positions as well as the residues found to be essential for the carrier activity, except Y202 which is located in the extramembrane loop IV-V, reside on the same face of transmembrane helix IV, probably lining part of a water-accessible crevice or channel between helices of the oxoglutarate carrier.

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The mitochondrial oxoglutarate carrier exchanges cytosolic malate for 2-oxoglutarate from the mitochondrial matrix. Orthologs of the carrier have a high degree of amino acid sequence conservation, meaning that it is impossible to identify residues important for function on the basis of this criterion alone. Therefore, each amino acid residue in the transmembrane alpha-helices H2 and H6 was replaced by a cysteine in a functional mitochondrial oxoglutarate carrier that was otherwise devoid of cysteine residues. The effects of the cysteine replacement and subsequent modification by sulfhydryl reagents on the initial uptake rate of 2-oxoglutarate were determined. The results were evaluated using a structural model of the oxoglutarate carrier. Residues involved in inter-helical and lipid bilayer interactions tolerate cysteine replacements or their modifications with little effect on transport activity. In contrast, the majority of cysteine substitutions in the aqueous cavity had a severe effect on transport activity. Residues important for function of the carrier cluster in three regions of the transporter. The first consists of residues in the [YWLF]- [KR]-G-X-X-P sequence motif, which is highly conserved in all members of the mitochondrial carrier family. The residues may fulfill a structural role as a helix breaker or a dynamic role as a hinge region for conformational changes during translocation. The second cluster of important residues can be found at the carboxy-terminal end of the even-numbered transmembrane alpha-helices at the cytoplasmic side of the carrier. Residues in H6 at the interface with H1 are the most sensitive to mutation and modification, and may be essential for folding of the carrier during biogenesis. The third cluster is at the midpoint of the membrane and consists of residues that are proposed to be involved in substrate binding.