(Received for publication, October 12, 1994; and in revised form, December 1, 1994)
From the
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
= 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
-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.
The tricarboxylate transport protein (i.e. citrate
transport protein) (CTP) ()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 [C]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
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.
Following incubation on ice, cells were harvested
by centrifugation at 5,000 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
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
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
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
g for 30 min, the resulting supernatant contained the
solubilized CTP.
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) .
Figure 2:
Lineweaver-Burk plot depicting the
dependence of the rate of citrate uptake on the external
[C]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
[C]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.
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
-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).
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 -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.
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U17503[GenBank].