©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
High Level Expression and Characterization of the Mitochondrial Citrate Transport Protein from the Yeast Saccharomyces cerevisiae(*)

(Received for publication, October 12, 1994; and in revised form, December 1, 1994)

Ronald S. Kaplan (1)(§) June A. Mayor (1) David A. Gremse (2) David O. Wood (3)

From the  (1)Departments of Pharmacology, (2)Pediatrics, and (3)Microbiology and Immunology, College of Medicine, University of South Alabama, Mobile, Alabama 36688

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The gene encoding the mitochondrial citrate transport protein (CTP) in the yeast Saccharomyces cerevisiae has been identified, and its protein product has been overexpressed in Escherichia coli. The expressed CTP accumulates in inclusion bodies and can be solubilized with sarkosyl. Approximately 25 mg of solubilized CTP at a purity of 75% is obtained per liter of E. coli culture. The function of the solubilized CTP has been reconstituted in a liposomal system where both its kinetic parameters (i.e.K = 0.36 mM and V(max) = 2.5 µmol/min/mg protein) and its substrate specificity have been determined. Notably, the yeast CTP displays a stricter specificity for tricarboxylates than do CTPs from higher eukaryotic organisms. Dot matrix analysis of the yeast CTP sequence indicates the presence of three homologous sequence domains (each approximately 100 residues in length), which are also related to domains in other CTPs. Thus, the yeast CTP displays the tripartite structure characteristic of other mitochondrial transporters. Alignment of the yeast CTP sequence with CTPs from other sources defines a consensus sequence that displays 89 positions of amino acid identity, as well as the more generalized mitochondrial transporter-associated sequence motif. Based on hydropathy analysis, the yeast CTP contains six putative membrane-spanning alpha-helices. Finally, Southern blot analysis indicates that the yeast genome contains a single gene encoding the mitochondrial CTP. Our data indicate that, based on both its structural and functional properties, the expressed yeast CTP can be assigned membership in the mitochondrial carrier family. The identification of the yeast CTP gene, and the expression and purification of large quantities of its protein product, pave the way for investigations into the roles of specific amino acids in the CTP translocation mechanism, as well as for the initiation of crystallization trials.


INTRODUCTION

The tricarboxylate transport protein (i.e. citrate transport protein) (CTP) (^1)from mammalian mitochondria catalyzes the efflux of citrate plus a proton across the mitochondrial inner membrane in exchange for another tricarboxylate-H, a dicarboxylate, or phosphoenolpyruvate (1) . Following diffusion through the outer membrane voltage-dependent anion channel, the resulting cytoplasmic citrate fuels both fatty acid and sterol biosyntheses and generates NAD for use in glycolysis(2, 3, 4, 5) . Because of its importance in bioenergetics, the CTP has been extensively characterized in higher eukaryotes. Thus, the CTP has been purified in reconstitutively active form(6, 7, 8) , kinetically characterized(9) , and its primary structure determined(10) . On the basis of both structural and functional considerations(10) , this protein has been assigned to the mitochondrial carrier family.

In contrast to the situation with higher eukaryotic cells, considerably less is known about the yeast mitochondrial CTP. Swelling studies (11) and direct measurement of [^14C]citrate uptake(12, 13) indicate that yeast mitochondria do contain a functional citrate carrier. The transporter has been kinetically characterized in mitochondria isolated from the yeast Saccharomyces cerevisiae where it displays a higher K value than observed for the mammalian CTP(13) . The reported V(max) values (12, 13) are extremely variable and appear to depend on the assay conditions employed. With respect to its substrate specificity, citrate, isocitrate, and to a considerably lesser extent malate serve as alternative substrates, whereas a variety of other dicarboxylates do not(13) . Persson and Srere (14) have obtained a partially purified preparation of the yeast CTP.

Recently, a sequence from S. cerevisiae appeared in GenBank (accession number X76053) (15) (obtained as part of the European Community yeast genome sequencing project), the deduced amino acid sequence of which displays a reasonably high identity (i.e. 37.7%) to the rat liver CTP. We therefore embarked on studies to express and characterize the protein encoded by this sequence. In the present paper, we report 1) the development of a procedure enabling the expression and purification of large quantities of the yeast CTP, 2) the functional characterization of the overexpressed yeast CTP following its incorporation into liposomal vesicles, and 3) a comparison of the structural properties of the yeast CTP with CTPs obtained from other sources. To our knowledge, this report presents the first information on the molecular properties of the yeast CTP and a definitive identification of its gene.


EXPERIMENTAL PROCEDURES

Construction of the Expression Plasmid

The yeast gene encoding the CTP corresponds to the reverse complement of nucleotides 29,856-30,755 of GenBank accession number X76053. The CTP gene plus an additional 66 nucleotides upstream (i.e. nucleotides 29,790-29,855) were amplified from total S. cerevisiae genomic DNA (Clontech; YNN 295) via PCR. Forward and reverse oligonucleotide primers were designed that corresponded to nucleotides 29,790-29,813 and 30,728-30,755, respectively. Linker sequences containing HindIII and BamHI or EcoRI and NdeI restriction sites were attached to the forward and reverse primers, respectively. PCR amplifications were conducted using the GeneAmp DNA amplification kit (Perkin-Elmer Corp.) in a Perkin Elmer GeneAmp PCR system 9600. The 100-µl reactions contained 15 ng of genomic DNA template and 1 µM primers. Following an initial incubation at 95 °C for 3.5 min, a three-step cycle that included denaturation at 94 °C (30 s), annealing at 60 °C (1 min), and extension at 72 °C (1 min, 40 s) was repeated a total of 32 times, followed by a final extension at 72 °C (7 min). The amplified DNA consisted of a single predominant 1-kilobase product, which was excised from a 2% agarose gel, purified using the Geneclean II kit (Bio 101, Inc.), restriction digested with NdeI and BamHI, extracted, and then purified from a 1.2% agarose gel as described above. Following digestion of pET-21a(+) plasmid DNA (Novagen) with NdeI and BamHI and purification of the linear vector fragment from a 1.2% agarose gel, the insert was directionally ligated into the pET plasmid, and the resulting construct was used to transform NovaBlue-competent cells (Novagen) according to the manufacturer's instructions. Transformants were screened for the presence of inserts via direct colony PCR (using the same primers as above) and restriction digestion of plasmid DNA that had been purified with the Wizard Minipreps DNA purification system (Promega). The resulting purified positive plasmids were then used to transform BL21(DE3)-competent cells (Novagen), the expression host, according to the manufacturer's instructions. Positive transformants were screened for inserts as described above.

Nucleotide Sequencing

The sequence of the cloned insert was determined using the Circumvent Thermal Cycle Dideoxy DNA sequencing kit (New England BioLabs). Plasmid DNA, purified as described above, was used as the template. T7 promoter and terminator primers were employed to sequence the ends of the insert. Internal primers were designed to enable sequencing of the remaining regions of each strand. A consensus sequence was developed based on data obtained from both strands of the insert. Partial sequencing of the PCR-amplified CTP DNA obtained from a series of separate reactions was accomplished employing either ``Genecleaned'' PCR product or PCR product that had been cloned into the pET plasmid (which was subsequently purified) as the template together with the primers described above. Nucleotide and amino acid sequence analyses were performed as previously described(10) .

Bacterial Expression and Isolation of the Yeast Citrate Transport Protein

Bacterial growth and induction of expression were carried out essentially according to the manufacturer's instructions (Novagen). Briefly, a single bacterial colony of Escherichia coli BL21(DE3) cells containing the target plasmid was used to inoculate 850 ml of LBC medium (1% bactotryptone, 0.5% yeast extract, 1% NaCl, pH 7.5, 50 µg of carbenicillin/ml). The culture was incubated with shaking (350 rpm) at 37 °C until a final absorbance at 600 nm of 0.6-0.7 was obtained. A 200-ml aliquot was then removed, placed on ice for 5 min, and harvested as described below. IPTG (1.0 mM) was then added to the remainder of the culture to induce expression of the CTP, and the incubation was continued for an additional 2 h. At this time, 200-ml aliquots were removed and placed on ice for 5 min.

Following incubation on ice, cells were harvested by centrifugation at 5,000 times g for 5 min (all centrifugations were conducted at 4 °C). The supernatants were discarded, and the sample pellets were stored on ice until all tubes were harvested. The cell pellets were resuspended in 20 ml of buffer A (50 mM Tris-HCl, 2 mM EDTA, pH 8.0). Lysozyme (100 µg/ml, freshly prepared in buffer A) and Triton X-100 (0.1%) were then added, and the cell suspensions were incubated for 15 min at 30 °C with occasional mixing. The resulting lysate was sonicated and centrifuged at 12,000 times g for 15 min, and the pellet was resuspended in 2 ml of buffer B (10 mM Tris-HCl, 0.1 mM EDTA, pH 7.0, 1.0 mM dithioerythritol) (16) and recentrifuged. The resulting pellet was resuspended in 2 ml of buffer B and fractionated by centrifugation at 131,000 times g for 4.5 h through a step gradient consisting of 12.4 ml of 40% (w/v) sucrose and 18.6 ml of 53% sucrose (16) (the sucrose solutions were prepared in buffer B). The inclusion body pellet was then resuspended in 30 ml of buffer B and centrifuged at 12,000 times g for 15 min. The CTP was extracted from the final inclusion body pellet by resuspension in 2.0 ml of ice-cold 1.2% (w/v) sarkosyl dissolved in buffer B(16) . Following centrifugation at 314,000 times g for 30 min, the resulting supernatant contained the solubilized CTP.

Functional Reconstitution of the Expressed Citrate Transport Protein

The solubilized CTP was reconstituted into preformed asolectin vesicles via the freeze-thaw-sonication technique. This established procedure, which has been used to characterize the function of the purified rat liver CTP(6) , was carried out as follows. Asolectin vesicles were prepared by bath sonication of dried asolectin (233.2 mg, Associated Concentrates) in 2.1 ml of buffer C (120 mM Hepes, 50 mM NaCl, 1 mM EDTA, pH 7.4) as previously described(17) . Solubilized CTP (5 µl) (12-15 µg of protein) was added to a mix consisting of 525 µl of asolectin vesicles, 60 µl of buffer C, 200 µl of buffer B, 120 µl of 400 mM citrate, 40 µl of 10% Triton X-114, and 45 µl of 1.2% sarkosyl in buffer B. This mixture was gently vortexed, rapidly frozen in liquid nitrogen, and then treated as previously detailed(6) .

Measurement of Reconstituted Citrate Transport

The preparation of BTC and its control buffer and a determination of the substrate specificity of the reconstituted CTP were carried out essentially as previously described(6) , except that transport was measured over a 20-s time interval. The kinetic parameters of the reconstituted yeast CTP were determined as follows. Proteoliposomes (50 µl) containing the solubilized expressed CTP were preincubated with 4 µl of either BTC control buffer (experimental tube) or 200 mM BTC (control tube) for 10 min. Transport was subsequently triggered via the addition of 25 µl of a given concentration of [1,5-^14C]citrate (external citrate ranged from 0.17 mM to 1.50 mM; specific radioactivity was approximately 2.1-4.8 times 10^4 dpm/nmol) (Amersham Corp.). Following a 10-s transport incubation, each experimental tube received 4 µl of 200 mM BTC, whereas each control tube received 4 µl of the BTC control buffer. The intraliposomal radiolabeled citrate was then separated by chromatography on Dowex columns (1.2 ml of resin equilibrated in buffer C)(6) , and the eluted (i.e. intraliposomal) radioactivity was quantified via liquid scintillation counting. The BTC-sensitive citrate transport rate was calculated by subtracting the control value from the experimental value obtained at each substrate concentration tested. Finally, determination of the rate of citrate uptake versus time profile indicated that measurements at 10 s yielded initial rate values.

Hybridization with Yeast Genomic DNA

Southern blot analysis of high molecular weight S. cerevisiae genomic DNA (5 µg/lane) (Clontech) was performed as previously described (10) except that hybridizations were carried out employing P-labeled yeast CTP gene (approximately 1.3 times 10^6 incorporated cpm/ml hybridization solution) as the probe and the dried nitrocellulose filter was exposed to x-ray film at -80 °C for approximately 20 min with two intensifying screens.

Miscellaneous

SDS-polyacrylamide gel electrophoresis was performed in a slab gel format using the buffer system of Laemmli (18) as previously described(6) . All samples, except the molecular weight standards, were precipitated with acetone prior to the electrophoretic separation(6) . The protein content of various fractions was determined as previously detailed(6) .


RESULTS AND DISCUSSION

High Level Expression of the Yeast Mitochondrial Citrate Transport Protein in E. coli and Subsequent Purification and Functional Reconstitution of the Expressed Transporter

To express the mitochondrial CTP from S. cerevisiae in E. coli, we pursued the following strategy. This strategy was generally based on the approach described by Fiermonte et al.(16) , which had enabled the successful expression of the mitochondrial alpha-ketoglutarate and ADP/ATP carriers. First, a yeast DNA sequence (accession number X76053)(15) , the deduced amino acid sequence of which displayed a reasonably high identity (i.e. 37.7%) to the rat liver mitochondrial CTP, was amplified by PCR, directionally cloned into the pET-21a(+) plasmid, and ultimately transformed into the expression host E. coli BL21(DE3). Second, following the initial growth of bacterial cells, CTP expression was induced by the addition of IPTG. Cells were harvested immediately prior to the IPTG addition, as well as 2 h later. Third, the inclusion body fraction was isolated, and the expressed CTP was extracted with the detergent sarkosyl. This detergent has been shown to successfully extract other mitochondrial transporters from inclusion bodies(16, 19) .

The SDS-PAGE profile that we obtained is depicted in Fig. 1. 2 h following induction with IPTG, a prominent protein band (apparent molecular mass 30-31 kDa) appears in the E. coli cell fraction (lane2) as well as in the inclusion body fraction isolated from these cells (lane3). This protein can be extracted from the inclusion bodies with sarkosyl (lane4). Scanning densitometric analysis of lane4 indicated that the solubilized expressed protein is approximately 75% pure. In contrast to these results, the SDS-PAGE profile of identical volumes of the same fractions obtained from cells harvested immediately prior to induction with IPTG indicates that negligible amounts of this protein band were present in the whole cell fraction (lane5), the isolated inclusion body fraction (lane6), or the final sarkosyl extract (lane7).


Figure 1: Coomassie-stained SDS-polyacrylamide gradient gel depicting expression of the yeast citrate transport protein in E. coli. Proteins were separated in a 4.5% polyacrylamide stacking gel followed by a 14-20% linear gradient gel. Lanes1 and 8, 1 µl of Bio-Rad SDS-PAGE low range molecular weight standards: phosphorylase b (97,400), serum albumin (66,200), ovalbumin (45,000), carbonic anhydrase (31,000), trypsin inhibitor (21,500), and lysozyme (14,400). Lanes2 and 5, 9.5 µl of E. coli cells harvested either 2 h following induction with IPTG (lane2) or immediately prior to the induction (lane5). Lanes3 and 6, 4 µl of the inclusion body fraction originating from cells harvested either 2 h following induction (lane3) or immediately prior to induction (lane6). Lanes4 and 7, 4 µl of the sarkosyl-solubilized inclusion body fraction originating from cells harvested either 2 h following induction (lane4) or immediately prior to induction (lane7).



To determine whether the expressed purified protein is in fact a citrate transporter, we incorporated the sarkosyl-solubilized protein into phospholipid vesicles and measured the ability of the resulting proteoliposomes to catalyze BTC-sensitive citrate/citrate exchange (i.e. the definitive property required of a mitochondrial CTP(1) ). We observed that the reconstituted, sarkosyl-solubilized inclusion body protein (isolated from IPTG-induced cells) catalyzed BTC-sensitive citrate/citrate exchange with a specific activity of 1.08 µmol/min/mg protein. In the absence of internal citrate, a BTC-sensitive citrate uptake rate of only 0.08 µmol/min/mg was observed. Thus, as has been observed with the rat liver mitochondrial CTP(7) , the reconstituted yeast transporter also displays a strict requirement for internal substrate. Most importantly, reconstitution of the sarkosyl-solubilized inclusion body fraction obtained from cells harvested immediately prior to induction with IPTG did not yield any detectable BTC-sensitive citrate/citrate exchange. In summary, the expression system described above enables the overexpression and subsequent purification of highly functional yeast mitochondrial CTP. Based on several separate inductions, we typically obtain approximately 25 mg of CTP at a purity of 75% per liter of E. coli culture.

Two additional points should be noted. First, analysis by SDS-PAGE indicated that, following cell lysis with lysozyme plus Triton X-100, most of the expressed CTP was found in the insoluble fraction. Second, our conclusion that the expressed CTP is localized within the inclusion body fraction rather than in an alternative membranous fraction is based primarily on our finding that, following sedimentation through 53% (w/v) sucrose, much of the expressed CTP was found to penetrate and even pellet through a 70% sucrose cushion. This would indicate a buoyant density similar to the value expected for inclusion bodies (20) .

Functional Characterization of the Expressed Yeast Citrate Transport Protein

The kinetic properties of the expressed yeast CTP were examined following incorporation of the sarkosyl-solubilized inclusion body CTP into phospholipid vesicles. Fig. 2depicts the dependence of the rate of citrate exchange on externally added [^14C]citrate in the presence of a constant internal concentration of citrate. A linear double-reciprocal plot is obtained, which yields K(m) and V(max) values of 0.36 mM and 2.48 µmol/min/mg protein, respectively. Interestingly, the K(m) value for the yeast CTP is slightly higher than the previously reported value of 0.13 mM for the purified rat liver mitochondrial CTP(9, 21) . The V(max) for the yeast CTP is similar to both the value reported for the reconstituted rat liver CTP (i.e. 1.9-2.0 µmol/min/mg protein using a similar assay system(9, 21) ) as well as to the value reported for a partially purified preparation of yeast CTP (i.e. 2.6 µmol/min/mg protein using a substantially different transport assay(14) ). It should be noted, however, that due to the absence of a high affinity specific molecular probe for the CTP, the proportion of reconstituted transporter that is in fact active is not known for any of the above preparations, thereby precluding the calculation of a meaningful molecular turnover number.


Figure 2: Lineweaver-Burk plot depicting the dependence of the rate of citrate uptake on the external [^14C]citrate concentration. The overexpressed yeast CTP was extracted from inclusion bodies with sarkosyl and incorporated into phospholipid vesicles in the presence of 48 mM citrate. Transport reactions were carried out for 10 s at 30 °C in the presence of 0.17-1.50 mM external citrate as described under ``Experimental Procedures.'' A best fit line was constructed based on linear regression analysis via the method of least squares. Data represent means of at least seven incubations ± S.E.



The substrate specificity of the reconstituted yeast CTP was examined by measuring the ability of a high concentration (20 mM) of various external anions to inhibit the [^14C]citrate/citrate exchange. As depicted in Table 1, tricarboxylates such as citrate (unlabeled) and isocitrate were effective inhibitors. Also, phosphoenolpyruvate had some inhibitory effect on the reconstituted CTP. In contrast, ADP, dicarboxylates, glutamate, phosphate, and pyruvate were ineffective inhibitors of the reconstituted transport. These results indicate that the reconstituted yeast CTP displays (i) a generally similar substrate specificity to that observed for this transporter in intact yeast mitochondria (13) and (ii) a significantly stricter specificity for tricarboxylates than has been observed for the CTP from higher eukaryotic organisms(1, 6, 7, 22) . For example, the reconstituted rat liver CTP transports tricarboxylates, malate, and phosphoenolpyruvate to similar extents(6, 7) . In summary, our data indicate that, based on functional considerations, the expressed CTP can be assigned membership in the mitochondrial carrier family.



Strain-dependent Mutations in the Sequence of the Yeast Citrate Transport Protein Gene and Its Protein Product

Following PCR amplification and cloning of the CTP gene into BL21(DE3) cells, its nucleotide sequence was determined (accession number U17503). We found the sequence to be identical to the published sequence (which was described as encoding a protein that is homologous to the mitochondrial uncoupling proteins(15) ) except for 5 base substitutions (i.e. G A, A T, A T, T G, and G T at open reading frame positions 102, 237, 289, 303, and 795, respectively). Two of these base changes resulted in changes in the deduced amino acid sequence (i.e. M97L and S101R). It is important to note that the published sequence corresponds to yeast strain S288C, whereas our studies utilized genomic DNA from yeast strain YNN 295. To determine whether the observed sequence differences represented strain-dependent mutations versus PCRdependent nucleotide misincorporations, we conducted the following studies. First, we sequenced the relevant regions of amplified CTP DNA (using genomic DNA from yeast strain YNN 295 as the template in the amplification reactions) (Clontech) obtained from six separate amplification reactions. In all cases, we found identical substitutions, thereby indicating that these changes did not arise from random misincorporation artifacts. Next, we amplified CTP DNA using genomic DNA obtained from yeast strain S288C (Promega, Novagen) as the template. The relevant regions of the CTP DNA product obtained from three separate amplification reactions were sequenced. In all cases, the CTP DNA sequence from strain S288C was identical to the published sequence. Based on these results, we conclude that (i) the sequence of the CTP gene from the two yeast strains differs at 5 positions, and these differences represent bona fide strain-dependent mutations rather than PCR artifacts and ii) the CTPs that result from these changes differ in deduced amino acid sequence at two positions.

Molecular Characteristics of the Yeast Citrate Transport Protein

As depicted in Table 2, the yeast CTP is comprised of 299 amino acids with a calculated molecular mass of 32.2 kDa. This molecular mass is similar to the values obtained for CTPs from other sources (Table 2) as well as to the values obtained for all mitochondrial transport proteins that have been purified(23) . Recently, Persson and Srere (14) have reported the partial purification of a yeast CTP with a molecular mass in the 50-60-kDa range. However, we speculate that the CTP in their preparation had dimerized either during the purification procedure and/or during the gel electrophoresis of their sample. It is known that mitochondrial transporters do in fact tend to dimerize under certain conditions(23, 24, 25) . The isoelectric point and the net charge of the yeast CTP are similar to the values observed for CTPs from other sources (Table 2) as well as to values for other mitochondrial transporters(10) . Finally, the yeast CTP is the most polar of the mitochondrial carriers, displaying a relatively high polarity value (i.e. 42.8%) for an integral membrane protein.



Similarity of the Yeast Citrate Transport Protein Sequence to Other Sequences

A search of the GenBank and Swiss-Prot data bases revealed that the yeast CTP displays the greatest similarity to the rat liver CTP (37.7% identity in a 289-amino acid overlap; accession number L12016(10) ), a putative CTP from Caenorhabditis elegans (36.6% identity in a 287-amino acid overlap; accession number P34519(26, 27) ), and the yeast ACR1 protein, potentially a mitochondrial carrier protein (32.0% identity in a 297-amino acid overlap; accession number Z25485(28) ). Comparison of the yeast CTP to other mitochondrial carriers indicated significant identity to the ADP/ATP translocase (i.e. 27.9% identity over 294 residues; accession number X62123), the uncoupling protein (i.e. 26.6% identity over 286 residues; accession number P25874), the alpha-ketoglutarate carrier (i.e. 25.1% identity over 287 residues; accession number P22292), and the phosphate carrier (i.e. 27.0% identity over 141 residues; accession number P12234).

Sequence Repeats within Mitochondrial Citrate Transporters

To identify potential homologous sequence domains both within the yeast CTP and between CTPs from various sources, a dot matrix analysis was performed. As depicted in Fig. 3, self-comparison of the yeast CTP at either moderate or high stringency (i.e.panelsA and B, respectively) indicates three homologous sequence domains, each approximately 100 amino acids in length. Thus, the yeast CTP displays the tripartite structure that is characteristic of other mitochondrial transporters(10, 29, 30, 31, 32, 33, 34, 35, 36) . Smallerdiagonals that are parallel to the maindiagonal reveal additional homology within each domain. As depicted in panelsC and D, pairwise comparisons between the yeast CTP and CTPs from rat liver and C. elegans, respectively, reveal that even greater homology exists between each of the three sequence domains present in CTPs from different organisms compared with the internal homology that exists between these domains within the yeast CTP (panelA). In summary, the data presented in Fig. 3demonstrate that the three sequence domains present in each CTP are interrelated, thereby suggesting a common genetic origin.


Figure 3: Dot-plot comparisons between mitochondrial citrate transporter sequences. The yeast CTP sequence was compared with itself (panelsA and B), the precursor form of the rat liver CTP (panelC), and a putative CTP from C. elegans (panelD). Transporter sequences noted on the xaxes in panelsC and D correspond to accession numbers P32089 and P34519(10, 26) , respectively. Comparisons were conducted using the Genetics Computer Group programs at either moderate stringency (i.e.panelsA, C, and D: window of 30 and stringency of 13) or high stringency (i.e.panelB: window of 30 and stringency of 17).



Fig. 4depicts an alignment between the yeast CTP, the rat liver CTP, and the putative CTP from C. elegans. The derived consensus sequence indicates 89 locations at which identical amino acids are found in all three sequences. It is noteworthy that the CTPs display considerably more homology with each other than with the sequences of other mitochondrial carriers, as evidenced by the fact that alignment with other carriers reveals little identity(10) . Thus, the CTP consensus sequence depicts a number of conserved domains that are unique to the CTP and presumably play specialized roles in the CTP translocation mechanism. The derived CTP signature sequence will facilitate the identification of other CTP sequences as well as provide a guide for future site-directed mutagenesis studies.


Figure 4: Alignment of mitochondrial citrate transporter sequences. The yeast mitochondrial CTP sequence was compared with the rat liver mitochondrial precursor CTP sequence and a putative mitochondrial CTP sequence from C. elegans (see Fig. 3legend for accession numbers). The three sequences were aligned using the Genetics Computer Group PileUp program, and a consensus sequence denoting positions of identity among the three sequences was derived. The dottedlines beneath the consensus sequence refer to the locations of putative membrane-spanning alpha-helices (I-VI) within the yeast CTP sequence. Numbersabove the sequences refer to locations within the yeast CTP sequence.



Several additional points are noteworthy. First, the CTP sequences contain the more generalized mitochondrial transport protein-associated motif Pro-X-(Asp/Glu)-X-(Val/Ile/Ala)-(Lys/Arg)-X-(Arg/Lys/Gln/Ala)-(Leu/Met/Phe/Ile), which repeats two to three times in each of the mitochondrial transporters of known function that have been sequenced(10, 29, 32, 33, 37) . Second, the sequence alignment depicted in Fig. 4suggests that the yeast CTP probably does not contain a presequence since its starting point aligns very closely (i.e. within 2 residues) to the known amino-terminal alanine of the mature rat liver CTP. Furthermore, the residues near the amino terminus of the yeast CTP do not display the characteristics typical of a mitochondrial presequence (i.e. a positively charged amphipathic domain that does not contain acidic residues)(38, 39) . In this respect, the yeast CTP is similar to the yeast mitochondrial phosphate transport protein, which is also unlikely to contain a cleavable presequence(36) . Finally, the carboxyl-terminal two-thirds of the CTP is somewhat more highly conserved than is the amino-terminal domain (i.e. 32% identity versus 25% identity, respectively).

Hydropathy Profile of the Yeast Mitochondrial Citrate Transport Protein and Comparison with Mitochondrial Citrate Transporters from Other Sources

The hydropathy profile depicted in Fig. 5indicates that the yeast CTP contains six domains that are capable of spanning the mitochondrial inner membrane as alpha-helices. This analysis is in general agreement with that reported by Holmstrom et al.(15) for the yeast chromosomal sequence. Similar profiles are seen with mitochondrial CTPs from other sources, as well as with all other mitochondrial transporters of known function that have been sequenced(10, 29, 30, 31, 32, 33, 34, 35, 36) . It is noteworthy that i) the hydropathy plot suggests that the carboxyl terminus of the yeast CTP is embedded within the mitochondrial inner membrane and ii) in both the yeast and the rat liver CTPs, putative helix IV displays a somewhat reduced hydrophobicity. The presence of one or more amphipathic membrane-spanning alpha-helices appears to be a general characteristic of the mitochondrial transporters(10, 29, 30, 31, 32, 33, 34, 35, 36) .


Figure 5: Hydropathy profiles of mitochondrial citrate transport proteins. Hydropathy values were calculated as previously described (10) employing a window of 11 residues. The horizontal line at -0.4 depicts the average hydropathy of 84 sequenced soluble proteins(43) . I-VI represent potential membrane-spanning alpha-helices.



Additional insight is obtained by combining the sequence alignment depicted in Fig. 4with the hydropathy profile depicted in Fig. 5. For example, the locations of amino acid sequence identity are uniformly distributed throughout the yeast CTP. Thus, 30% of the putative transmembrane residues are identical, and 29% of the hydrophilic extramembranous residues are identical. In a hypothetical model consisting of six transmembrane domains connected by five hydrophilic extramembranous loops, it is interesting to note that the three hydrophilic loops connecting helices I-II, III-IV, and V-VI would all reside within the same aqueous compartment and display considerably more net positive charge (i.e. +5, +4, and +3, respectively) than do the two hydrophilic loops that connect helices II-III and IV-V and reside within the opposite aqueous compartment (i.e. -1 and no net charge, respectively). The mechanistic consequence of this charge asymmetry is not known at present. Finally, the data presented in Fig. 3Fig. 4Fig. 5and Table 2clearly indicate that, based on structural considerations, the expressed yeast CTP can be assigned to the mitochondrial carrier family.

Number of Genes Encoding the Yeast Mitochondrial Citrate Transport Protein

Southern hybridization, carried out under conditions of high stringency, was used to determine the number of sequences in the yeast genome that are closely related to the mitochondrial CTP. The results from these studies are presented in Fig. 6and indicate that with each of the four restriction enzymes tested, a single strongly hybridizing band was detected. Thus, we conclude that the yeast genome contains a single gene encoding the CTP and that other closely related sequences are not present. This situation is analogous to that observed with the yeast mitochondrial phosphate transporter, which is likely encoded by a single gene (36) but differs from that observed with the yeast ADP/ATP translocase, which is encoded by multiple genes(40, 41, 42) .


Figure 6: Hybridization of restriction digests of S. cerevisiae genomic DNA with a DNA probe encoding the yeast CTP. Genomic DNA from the yeast S. cerevisiae was digested to completion with EcoRI (E), HindIII (H), KpnI (K), and XbaI (X). Following blotting to nitrocellulose, the digests were probed with the PCR-amplified yeast CTP gene. The positions of the DNA markers are depicted at the left.



In conclusion, these studies have resulted in the identification of the yeast gene encoding the mitochondrial CTP, high level expression and purification of the yeast CTP gene product, and structural and functional characterization of the highly purified expressed yeast CTP. These developments will now permit site-directed mutagenesis to be utilized to ascertain the roles of specific residues in the CTP translocation mechanism, as well as the initiation of crystallization trials.


FOOTNOTES

*
This work was supported by National Science Foundation Grant MCB-9219387 (to R. S. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U17503[GenBank].

§
To whom correspondence should be addressed. Tel.: 334-460-6057; Fax: 334-460-6798.

(^1)
The abbreviations used are: CTP, citrate transport protein; BTC, 1,2,3-benzenetricarboxylate; IPTG, isopropyl-1-thio-beta-D-galactopyranoside; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.


ACKNOWLEDGEMENTS

We thank Dr. David R. Nelson for drawing attention to the presence of a sequence in GenBank (accession number X76053) that displayed significant similarity to the rat liver CTP sequence and for other helpful suggestions. We also thank Brenda Dean for expert technical assistance.


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