Ronald S. Kaplan

Structure and function of mitochondrial anion transport proteins.

The mitochondrial anion transporters confer a highly selective permeability upon the mitochondrial inner membrane. To date, at least fourteen anion transporter activities have been clearly identified (see Table 1). These carriers catalyze a high magnitude flux across the inner membrane and as such occupy a particularly prominent position within eukaryotic cell intermediary metabolism. For example, the exchange of cytoplasmic ADP for mitochondrial ATP on the adenine nucleotide transporter, as well as the import of phosphate on the phosphate carrier, are essential in order to provide the cell with ATP made via oxidative phosphorylation. Citrate efflux from mitochondria on the citrate transporter provides the cytoplasm with a carbon source that supports fatty acid and sterol biosyntheses. Additionally, this efflux likely provides a supply of NAD (via the concerted action of ATP-citrate lyase and malate dehydrogenase) for use by the glycolytic pathway. Malate efflux from mitochondria on the dicarboxylate carrier represents a required step in the gluconeogenic pathway. Alternatively, in yeast this carrier may catalyze the influx of malate, thereby providing an anaplerotic function. Pyruvate influx into mitochondria via the pyruvate transporter is a required step for: (i) the complete oxidation of glucose and amino acids (thereby providing the energy source for a substantial portion of the cell’s ATP); and (ii) the supply of carbon precursor for the gluconeogenic as well as the triacylglycerol and sterol biosynthetic pathways. Finally, the a-ketoglutarate and the aspartate/ glutamate carriers play essential roles in the malate/ aspartate shuttle, as well as in gluconeogenesis. The well-established anion transporters and their metabolic importance are listed in Table 1. The progress to date in the study of these transporters is summarized in Table 2. Because of the importance of the mitochondrial anion transporters in physiology and disease, considerable effort has been expended to extend our understanding of these carriers to the molecular level. This review will summarize the current state of knowledge concerning the molecular basis underlying the functioning of the major mitochondrial anion transport proteins. Emphasis will be placed on the structure-function relationships that have been elucidated to date, and on the more recent approaches that have been developed which will likely guide future efforts. Since a goal of this review is to be brief, I will primarily focus on developments during the last 5 years and citations to literature will be selective. For earlier information, as well as information regarding other (i.e., the neutral and cationic) metabolite mitochondrial transporters, the reader is referred to several insightful earlier reviews (LaNoue & Schoolwerth, 1979; LaNoue & Schoolwerth, 1984; Kramer & Palmieri, 1989; Kramer & Palmieri, 1992; Walker, 1992). Also, detailed information on mitochondrial carrier sequences Correspondence to: R.S. Kaplan

Sensitive protein assay in presence of high levels of lipid.

Publisher Summary This chapter describes a method that is capable of accurately measuring low amounts of a protein in the presence of very high levels of lipid. This procedure was developed from the amido black 10 B methods of Schaffner and Weissmann and Newman et al. and incorporates several critical modifications that enable an assay to be performed with lipid-containing samples without any interference. One approach has been to remove an interfering lipid by extraction with organic solvents. However, because certain proteins display a limited solubility in such solvents, this strategy often fails. Another widely used approach involves the inclusion of sodium dodecyl sulfate (SDS) in a modified Lowry procedure to reduce lipid (and detergent) interference. As oxidized lipid continues to react to produce a substantial amount of color in the Lowry assay and as most lipid samples are partially oxidized, this procedure is not suitable for the accurate measurements of a protein in samples containing excess of lipid.

Streptozotocin-induced alterations in the levels of functional mitochondrial anion transport proteins

The effect of streptozotocin-induced diabetes on the levels of functional mitochondrial anion transport proteins has been determined. The experimental approach utilized for these studies consisted of the extraction of each of four mitochondrial anion transport proteins from rat liver mitoplasts (isolated from diabetic and control animals) with the nonionic detergent Triton X-114, followed by the functional reconstitution of each transporter in a liposomal system via the freeze-thaw-sonication technique. This approach permitted the quantification of transporter function without the complications that occur when such measurements are carried out with intact mitochondria (or mitoplasts). We found that experimental diabetes caused an increase in the extractable and reconstitutable specific (and total) transport activities of the pyruvate and dicarboxylate transporters, a decrease in the activity of the citrate transporter, and no significant change in the activity of the phosphate transporter relative to control values. An examination of the time course of the appearance of changes in the reconstitutable activities of the pyruvate and citrate transporters following the injection of streptozotocin revealed differences. Thus, whereas the activity of the pyruvate transporter displayed the most pronounced increase (193%) 1 week following streptozotocin injection and then subsequently declined from this peak and plateaued at later times (99% and 96% increases at 3 and 8 weeks, respectively), the activity of the citrate transporter progressively decreased with time (31-51% decreases at 1-8 weeks). We suggest that the observed diabetes-induced changes in mitochondrial anion transporter function are predictable on the basis of diabetes-induced alterations in the activities of enzymes that constitute metabolic pathways to which these transporters either supply substrate or remove product. Furthermore, we speculate that mitochondrial anion transport proteins may be regulated in coordination with the enzymes of such associated metabolic pathways.

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.