June A. Mayor

Identification of a Novel Gene Encoding the Yeast Mitochondrial Dicarboxylate Transport Protein via Overexpression, Purification, and Characterization of Its Protein Product

A gene encoding the mitochondrial dicarboxylate transport protein (DTP) has been identified for the first time from any organism. Our strategy involved overexpression of putative mitochondrial transporter genes, selected based on analysis of the yeast genome, followed by purification and functional reconstitution of the resulting protein products. The DTP gene from the yeast Saccharomyces cerevisiae encodes a 298-residue basic protein which, in common with other mitochondrial anion transporters of known sequence and function, displays the mitochondrial transporter signature motif, three homologous 100-amino acid sequence domains, and six predicted membrane-spanning regions. The product of this gene has been abundantly expressed in Escherichia coli where it accumulates in inclusion bodies. Upon solubilization of the overexpressed DTP from isolated inclusion bodies with Sarkosyl, 28 mg of DTP was obtained per liter of E. coli culture at a purity of 75%. The purified, overexpressed DTP was then reconstituted in phospholipid vesicles where both its kinetic properties (i.e. Km = 1.55 mM and Vmax = 3.0 μmol/min/mg protein) and its substrate specificity were determined. The intraliposomal substrates malonate, malate, succinate, and phosphate effectively supported [14C]malonate uptake, whereas other anions tested did not. External substrate competition studies revealed a similar specificity profile. Inhibitor studies indicated that the reconstituted transporter was sensitive to inhibition by n-butylmalonate, p-chloromercuribenzoate, mersalyl, and to a lesser extent pyridoxal 5′-phosphate but was insensitive to N-ethylmaleimide and selective inhibitors of other mitochondrial anion transporters. In combination, the above findings indicate that the identified gene encodes a mitochondrial transport protein which upon overexpression and reconstitution displays functional properties that are virtually identical to those of the native mitochondrial dicarboxylate transport system. In conclusion, the present investigation has resulted in identification of a gene encoding the mitochondrial DTP and thus eliminates a major impediment to molecular studies with this metabolically important transporter. Based on both structural and functional considerations, the yeast DTP is assignable to the mitochondrial carrier family. Additionally, the development of a procedure that enables the expression and isolation of large quantities of functional DTP provides the foundation for comprehensive investigations into the structure/function relationships within this transporter via site-directed mutagenesis, as well as for the initiation of crystallization trials.

The Yeast Mitochondrial Citrate Transport Protein PROBING THE ROLES OF CYSTEINES, Arg181, AND Arg189 IN TRANSPORTER FUNCTION

Utilizing site-directed mutagenesis in combination with chemical modification of mutated residues, we have studied the roles of cysteine and arginine residues in the mitochondrial citrate transport protein (CTP) from Saccharomyces cerevisiae. Our strategy consisted of the sequential replacement of each of the four endogenous cysteine residues with Ser or in the case of Cys73 with Val. Wild-type and mutated forms of the CTP were overexpressed in Escherichia coli, purified, and reconstituted in phospholipid vesicles. During the sequential replacement of each Cys, the effects of both hydrophilic and hydrophobic sulfhydryl reagents were examined. The data indicate that Cys73 and Cys256 are primarily responsible for inhibition of the wild-type CTP by hydrophilic sulfhydryl reagents. Experiments conducted with triple Cys replacement mutants (i.e. Cys192 being the only remaining Cys) indicated that sulfhydryl reagents no longer inhibit but in fact stimulate CTP function 2–3-fold. Following the simultaneous replacement of all four endogenous Cys, the functional properties of the resulting Cys-less CTP were shown to be quite similar to those of the wild-type protein. Finally, utilizing the Cys-less CTP as a template, the roles of Arg181 and Arg189, two positively charged residues located within transmembrane domain IV, in CTP function were examined. Replacement of either residue with a Cys abolishes function, whereas replacement with a Lys or a Cys that is subsequently covalently modified with (2-aminoethyl)methanethiosulfonate hydrobromide, a reagent that restores positive charge at this site, supports CTP function. The results clearly show that positive charge at these two positions is essential for CTP function, although the chemistry of the guanidinium residue is not. Finally, these studies: (i) definitely demonstrate that Cys residues do not play an important role in the mechanism of the CTP; (ii) prove the utility of the Cys-less CTP for studying structure/function relationships within this metabolically important protein; and (iii) have led to the hypothesis that the polar face of α-helical transmembrane domain IV, within which Arg181, Arg189, and Cys192 are located, constitutes an essential portion of the citrate translocation pathway through the membrane.

Oligomeric State of Wild-Type and Cysteine-Less Yeast Mitochondrial Citrate Transport Proteins

Experiments have been conducted to determine the oligomeric state of the mitochondrial citratetransport protein (CTP) from the yeast Saccharomyces cerevisiae. Both wild-type andcysteine-less (Cys-less) CTPs were overexpressed in E. coli and solubilized with sarkosyl. The purity ofthe solubilized material is approximately 75%. Upon incorporation into phospholipid vesicles, ahigh specific transport activity is obtained with both the wild-type and Cys-less CTPs, therebydemonstrating the structural and functional integrity of the preparations. Two independentapproaches were utilized to determine native molecular weight. First, CTP molecular weightwas determined via nondenaturing size-exclusion chromatography. With this methodology weobtained molecular weight values of 70,961 and 70,118 for the wild-type and Cys-less CTPs,respectively. Second, charge-shift native gel electrophoresis was carried out utilizing a lowconcentration of the negatively charged detergent sarkosyl, which served to both impart acharge shift to the CTP and the protein standards, as well as to promote protein solubility.Via the second method, we obtained molecular weight values of 69,122 and 74,911 forthe wild-type and Cys-less CTPs, respectively. Both methods clearly indicate that followingsolubilization, the wild-type and the Cys-less CTPs exist exclusively as dimers. Furthermore,disulfide bonds are not required for either dimer formation or stabilization. The dimericstate of the CTP has important implications for the structural basis underlying the CTPtranslocation mechanism.

Structure, function and regulation of the tricarboxylate transport protein from rat liver mitochondria

Recent progress is summarized on the structure, function, and regulation of the tricarboxylate (i.e., citrate) transport protein (CTP) from the rat liver mitochondrial inner membrane. The transporter has been purified and its reconstituted function characterized. A cDNA clone encoding the CTP has been isolated and sequenced, thus enabling a deduction of the complete amino acid sequence of this 32.6 kDa transport protein. Dot matrix analysis and sequence alignment indicate that based on structural considerations the CTP can be assigned to the mitochondrial carrier family. Hydropathy analysis of the transporter sequence indicates six putative membrane-spanning α-helices and has permitted the development of an initial model for the topography of the CTP within the inner membrane. The questions as to whether more than one gene encodes the CTP and whether more than one isoform is expressed remain unanswered at this time. Studies documenting a diabetes-induced alteration in the function of several mitochondrial anion transporters, which can be reversed by treatment with insulin, provide a physiologically/pathologically relevant experimental system for studying the molecular mechanism(s) by which mitochondrial transporters are regulated. Potential future research directions are discussed.

Identification of the Substrate Binding Sites within the Yeast Mitochondrial Citrate Transport Protein

The objective of the present investigation was to identify the substrate binding site(s) within the yeast mitochondrial citrate transport protein (CTP). Our strategy involved kinetically characterizing 30 single-Cys CTP mutants that we had previously constructed based on their hypothesized importance in the structure-based mechanism of this carrier. As part of these studies, a modified transport assay was developed that permitted, for the first time, the accurate determination of Km values that were elevated >100-fold compared with the Cys-less control value. We identified 10 single-Cys CTP mutants that displayed sharply elevated Km values (i.e. 5 to >300-fold). Each of these mutants displayed Vmax values that were reduced by ≥98% and resultant catalytic efficiencies that were reduced by ≥99.9%. Importantly, superposition of this functional data onto the three-dimensional homology-modeled CTP structure, which we previously had developed, revealed that nine of these ten residues form two topographically distinct clusters. Additional modeling showed that: (i) each cluster is capable of forming numerous hydrogen bonds with citrate and (ii) the two clusters are sufficiently distant from one another such that citrate is unlikely to interact with all of these residues at the same time. We deduced from these findings that the CTP contains at least two citrate binding sites per monomer, which are located at increasing depths within the translocation pathway. The identification of these sites, combined with an initial assessment of the citrate-amino acid side-chain interactions that may occur at these sites, substantially extends our understanding of CTP functioning at the molecular level.

The effect of insulin supplementation on diabetes-induced alterations in the extractable levels of functional mitochondrial anion transport proteins☆

The effect of insulin supplementation on diabetes-induced alterations in the levels of functional mitochondrial anion transport proteins has been determined. The experimental approach consisted of the extraction of the pyruvate, dicarboxylate, and citrate transport proteins from the mitochondrial inner membrane with Triton X-114 using rat liver mitoplasts (prepared from control, diabetic, or insulin-supplemented diabetic animals) as the starting material, followed by the reconstitution of the function of each transporter in a proteoliposomal system. This experimental strategy permitted the quantification of the functional levels of these three transporters in the absence of the complications that arise when such measurements are carried out with intact mitochondria (or mitoplasts). We found that treatment of diabetic rats (i.e., animals that were injected with streptozotocin 3 weeks earlier) on a daily basis with insulin for 3 weeks resulted in a reversal of the diabetes-induced (a) increase in the extractable and reconstitutable total (and specific) transport activities of the pyruvate and dicarboxylate transporters and (b) decrease in the activity of the citrate transporter. These findings indicate that diabetes-induced alterations in the functional levels of mitochondrial anion transport proteins are a direct consequence of the insulin insufficiency that characterizes this disease. Furthermore, this study provides the first demonstration that insulin participates in the regulation of the functional levels of liver mitochondrial anion transport proteins.

Functional levels of mitochondrial anion transport proteins in non-insulin-dependent diabetes mellitus

The effect of non-insulin-dependent diabetes mellitus (i.e., NIDDM; type 2 diabetes) on the levels of functional mitochondrial anion transport proteins has been determined utilizing a chemically-induced neonatal model of NIDDM. We hypothesized that moderate insulin deficiency exacerbated by the insulin resistance, which is characteristic of NIDDM, would cause changes in mitochondrial anion transporter function that were similar to those we have previously shown to occur in insulin-dependent diabetes mellitus (i.e., IDDM; type 1 diabetes) (Arch. Biochem. Biophys. 280: 181–191, 1990). Our experimental approach consisted of the extraction of the pyruvate, dicarboxylate and citrate transport proteins from the mitochondrial inner membrane with Triton X-114 using rat liver mitoplasts (prepared from diabetic and control animals) as the starting material, followed by the functional reconstitution of each transporter in a proteoliposomal system. This strategy permitted the quantification of the functional levels of these three transporters in the absence of the complications that arise when such measurements are carried out with intact mitochondria (or mitoplasts). We found that experimental NIDDM did not cause significant changes in the extractable and reconstitutable specific (and total) transport activities of the pyruvate, dicarboxylate, and citrate transporters. These results are in marked contrast to our previous findings obtained using rats with IDDM and negated our hypothesis. The present results, in combination with our earlier findings, allow us to conclude that insulin plays an important role in the regulation of mitochondrial anion transporter function. Accordingly, in this model of NIDDM, where the level of insulin is not profoundly deficient, transporter function is unaltered, whereas in IDDM, where a profound insulinopenia exists, transporter function is altered. Furthermore, the present studies suggest that in the neonatal model of NIDDM the three mitochondrial transporters investigated are neither affected by, nor are they the sites of the well documented hepatic post-receptor insulin resistance which is characteristic of this disease.

The yeast mitochondrial citrate transport protein: molecular determinants of its substrate specificity.

The objective of this study was to identify the role of individual amino acid residues in determining the substrate specificity of the yeast mitochondrial citrate transport protein (CTP). Previously, we showed that the CTP contains at least two substrate-binding sites. In this study, utilizing the overexpressed, single-Cys CTP-binding site variants that were functionally reconstituted in liposomes, we examined CTP specificity from both its external and internal surfaces. Upon mutation of residues comprising the more external site, the CTP becomes less selective for citrate with numerous external anions able to effectively inhibit [14C]citrate/citrate exchange. Thus, the site 1 variants assume the binding characteristics of a nonspecific anion carrier. Comparison of [14C]citrate uptake in the presence of various internal anions versus water revealed that, with the exception of the R189C mutant, the other site 1 variants showed substantial uniport activity relative to exchange. Upon mutation of residues comprising site 2, we observed two types of effects. The K37C mutant displayed a markedly enhanced selectivity for external citrate. In contrast, the other site 2 mutants displayed varying degrees of relaxed selectivity for external citrate. Examination of internal substrates revealed that, in contrast to the control transporter, the R181C variant exclusively functioned as a uniporter. This study provides the first functional information on the role of specific binding site residues in determining mitochondrial transporter substrate selectivity. We interpret our findings in the context of our homology-modeled CTP as it cycles between the outward-facing, occluded, and inward-facing states.

Bacterial Overexpression of Putative Yeast Mitochondrial Transport Proteins

Thirty-two genes have been identified within the genome of the yeast Saccharomyces cerevisiae which putatively encode mitochondrial transport proteins. We have attempted to overexpress a subset of these genes, namely those which encode mitochondrial transporters of unknown function, and have succeeded in overexpressing 19 of these genes. The overexpressed proteins were then isolated and tested for five well-characterized reconstituted transport activities (i.e., the transport of citrate, dicarboxylates, pyruvate, camitine, and aspartate). Utilizing this approach, we have clearly identified the yeast mitochondrial dicarboxylate transport protein, as well as two additional lower-magnitude transport functions (i.e., tricarboxylate and dicarboxylate transport activities). The implications of these results and the considerations relevant to this approach are discussed.

The yeast mitochondrial citrate transport protein: identification of the Lysine residues responsible for inhibition mediated by Pyridoxal 5′-phosphate

The present investigation identifies the molecular basis for the well-documented inhibition of the mitochondrial inner membrane citrate transport protein (CTP) function by the lysine-selective reagent pyridoxal 5′-phosphate. Kinetic analysis indicates that PLP is a linear mixed inhibitor of the Cys-less CTP, with a predominantly competitive component. We have previously concluded that the CTP contains at least two substrate binding sites which are located at increasing depths within the substrate translocation pathway and which contain key lysine residues. In the present investigation, the roles of Lys-83 in substrate binding site one, Lys-37 and Lys-239 in substrate binding site two, and four other off-pathway lysines in conferring PLP-inhibition of transport was determined by functional characterization of seven lysine to cysteine substitution mutants. We observed that replacement of Lys-83 with cysteine resulted in a 78% loss of the PLP-mediated inhibition of CTP function. In contrast, replacement of either Lys-37 or Lys-239 with cysteine caused a modest reduction in the inhibition caused by PLP (i.e., 31% and 20% loss of inhibition, respectively). Interestingly, these losses of PLP-mediated inhibition could be rescued by covalent modification of each cysteine with MTSEA, a reagent that adds a lysine-like moiety (i.e. SCH2CH2NH3+) to the cysteine sulfhydryl group. Importantly, the replacement of non-binding site lysines (i.e., Lys-45, Lys-48, Lys-134, Lys-141) with cysteine resulted in little change in the PLP inhibition. Based upon these results, we conducted docking calculations with the CTP structural model leading to the development of a physical binding model for PLP. In combination, our data support the conclusion that PLP exerts its main inhibitory effect by binding to residues located within the two substrate binding sites of the CTP, with Lys-83 being the primary determinant of the total PLP effect since the replacement of this single lysine abolishes nearly all of the observed inhibition by PLP.