The Solute Carrier (SLC) Complement of the Human Genome: Phylogenetic Classiﬁcation Reveals Four Major Families

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FEBS Letters 582 (2008) 3811–3816

The solute carrier (SLC) complement of the human genome: Phylogenetic classiﬁcation reveals four major families

Robert Fredriksson*, Karl J.V. Nordstro¨m, Olga Stephansson, Maria G.A. Ha¨gglund, Helgi B. Schio¨th Department of Neuroscience, Unit of Functional Pharmacology, Uppsala University, BMC, Uppsala SE 75124, Sweden

Received 4 September 2008; revised 3 October 2008; accepted 10 October 2008

Available online 21 October 2008

Edited by Takashi Gojobori

the plasma membrane while others are specifically localized Abstract Solute carriers (SLCs) is the largest group of transporters, embracing transporters for inorganic ions, amino acids, in mitochondria [11], synaptic vesicles [13] or peroxisomes [14]. neurotransmitters, sugars, purines and fatty acids among other The SLC family ranks among the largest families of mem- substrates. We mined the finished assembly of the human genome brane proteins in human together with G protein-coupled using Hidden Markov Models (HMMs) obtaining a total of 384 receptors (GPCRs) [15], voltage gated ion channels [1] and unique SLC sequences. Detailed clustering and phylogenetic tyrosine kinase receptors [16]. The repertoire and phylogenetic analysis of the entire SLC family showed that 15 of the families relationships of all human proteins within most of the major place into four large phylogenetic clusters with the largest con- families of membrane proteins, except the SLCs, have previ- taining eight SLC families, suggesting that many of the distinct ously been described. The GPCR family is by far the largest families of SLCs have a common evolutionary origin. This study with about 800 members [17,18] followed by voltage gated represents the first overall genomic roadmap of the SLCs provid- ion channels with 143 members [1] and 105 transmembrane ing large sequence sets and clarifies the phylogenetic relationships among the families of the second largest group of protein kinases [16]. Despite the SLC family being the second membrane proteins. largest family of membrane proteins, the entire SLC content of Ó 2008 Federation of European Biochemical Societies. Pub- a single vertebrate genome has not been mined and analyzed lished by Elsevier B.V. All rights reserved. systematically. One reason for this is that the phylogenetic relationship between the SLCs is far more complex than for Keywords: Transporter; Membrane bound; SLC; Phylogeny; the other large families of membrane proteins, with a large Repertoire; Orphan number of families with unclear relationships regarding structural properties or evolutionary origin. Another reason is that these genes have tremendously complex genomic structure, generally with a high number of introns. In this paper, we mined all genes from the finished assembly 1. Introduction of the human genome [19] using custom made sequence Hid- den Markov Models (HMMs), and obtained a total of 384 un- Transporter proteins can be grouped in various classes ique SLC sequences. We performed a detailed phylogenetic where the most important are ion channels [1], ABC transport- analysis of the entire SLC family and found that 15 of the fam- ers [2], water channels [3], pumps such as the sodium potas- ilies place into four large phylogenetic clusters with the largest sium pump [4] and the solute carriers (SLCs). The SLCs are containing seven SLC families. This indicates that some of the by far the largest group of transporters. In 2003 a list of 298 distinct families of SLCs have a common evolutionary origin. SLCs in 43 families were presented [5] based on the HUGO assembly of sequences. Three additional families, SLC44– SLC46 containing in total 12 genes, have since been described 2. Results [6–8]. Moreover, the number of members from the other SLC families has continued to increase and new members have been We made a comprehensive screening of the four largest identified from for example the SLC5 [9], SLC6 [10], SLC25 available collections of human mRNA and cDNA sequences, [11], and SLC35 families [12], in total approximately 50 mem- RefSeq, Ensembl peptides, Ensembl ab initio and the human bers, although this has not been summarized systematically. subset of the Mammalian Gene Collection (MGC), using se- The members of the SLC families have varied biochemical quence HMMs. We built 46 HMMs (see Supplementary data properties with some being coupled transporters, some being 1), one for each SLC family. All models were screened on all exchangers, often driven by the cellular sodium gradient, and sequence sets and a non-redundant SLC dataset was con- some being passive transporters, only translocating molecules structed by first aligning all SLC sequences with the human along their concentration gradient. Their cellular localization genome sequence and then removing all entries that align to also varies with the majority of the transporters localized to the same position. Thereafter, all sequences with higher similarity to any non-SLC protein in the RefSeq database were removed. This resulted in a dataset of 384 sequences of which 19 were not previously described as SLCs. We submitted these se- *Corresponding author. Fax: +46 18 51 15 40. E-mail address: [email protected] (R. Fredriksson). quences to the HUGO nomenclature committee (HGNC) and

0014-5793/$34.00 Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2008.10.016 3812 R. Fredriksson et al. / FEBS Letters 582 (2008) 3811–3816

HGNC confirmed that these genes were not previously anno- Table 1 tated and subsequently assigned symbols according to their Summary of the SLC families in the human genome. nomenclature rules and made them public with our consent. SLC-family Number Group Some of these transporters have subsequently been function- SLC1 7 – ally characterized by others [20,21]. SLC2 15 a We grouped the sequences from different SLC families into SLC3 2 – larger phylogenetic hierarchies. The families that had members SLC4 10 – SLC5 12 – with similarity (see Supplementary Methods) to members from SLC6 20 – other families were merged and analyzed phylogenetically with SLC7 13 c the maximum parsimony method to form the a-, b-, c- and d- SLC8 3 d groups, see below. We thereafter used the topology from the SLC9 11 – parsimony analysis as a basis for maximum likelihood SLC10 7 – SLC11 2 – branch-length mapping, to produce the phylograms seen in SLC12 9 c Fig. 1. The properties of the alignments that the maximum SLC13 6 – parsimony trees were based on were further evaluated consid- SLC14 2 – ering information content, expressed as the number of infor- SLC15 5 – SLC16 15 a mative sites. All alignments are presented in Supplementary SLC17 9 a data 4. SLC18 3 a We found that 15 of the previously described SLC families SLC19 3 – as well as the SV2 family (Fig. 1, Table 1) fall into four main SLC20 2 – phylogenetic clusters, which we term the a-, b-, c- and d- SLC21 11 – SLC22 28 a groups to distinguish them from the previously designed fam- SLC23 4 – ilies of SLCs. This shows a phylogenetic grouping of the SLC SLC24 6 d families that are much wider than previously described. We SLC25 46 – also used Neighbor Joining (NJ) to verify our topologies for SLC26 10 – SLC27 6 – the a-, b-, c- and d-groups and these trees are available as Sup- SLC28 3 – plementary data 2. SLC29 5 – In Fig. 1 we show these main phylogenetic clusters. The a- SLC30 10 – group contains seven families of SLCs and a cluster of proteins SLC31 2 – termed Synaptic Vesicle 2 proteins (SV2) [22] which we have SLC32 1 b SLC33 1 – previously shown to be phylogenetically related to the SLC34 3 – SLC22 family [23]. The alignments behind the a-tree is 1137 SLC35 23 – amino acid long with 50.1% percent of these being parsimoni- SLC36 4 b ous informative. The topology of the Neighbor Joining tree SLC37 4 a SLC38 11 b (see Supplementary data 2) is similar. SLC39 14 – The other families that place in the a-group are SLC17 SLC40 1 – (vesicular glutamate transporters), SLC18 (vesicular mono- SLC41 3 – amine transporters), SLC16 (transporters for among others SLC42 3 – lactate), SLC22 (transporters for organic ions) and finally SLC43 3 – SLC44 5 – SLC37 and SLC2 which both transport sugar compounds. SLC45 4 – The a-group also contains six sequences with relatively long SLC46 3 a branches. Three of these have previously been shown to be related to the SLC22 family [23,24], two (MFSD9 and SV2 5 a C6ORF192) belongs to the MFSD proteins and are first de- Other 9 – Total 384 – scribed here with names designated according to advice from the HGNC and one is the likely human homolog of the pro- karyotic tetracycline transporter (TETRAN). We also found The c-group contains the SLC7 and SLC12 families and we three new genes that clearly place into well-known phyloge- identified one new member from the SLC7 family, named netic clusters, two in the SLC16 family (SLC16A12 and SLC7A14. The topology is based on an alignment with 629 SLC16A15) and one (SLC17A9) in the SLC17 family and all amino acids, where 71% are parsimoniously informative. The three of these have been assigned names by the HGNC. node joining the two families has a bootstrap support of 911 The second largest cluster is the b-group containing three (see Supplementary data 3). SLC7A14 groups closely with families (SLC32, SLC36 and SLC38) of amino acid transport- SLC7A1-4 among which SLC7A4 has an unknown substrate ers. The topology is based on an alignment with 548 positions while SLC7A1 and A3 all transport Cationic LL-amino acids. of which 68% are parsimoniously informative and the deep All SLC7 family transporters with known substrates carry cat- nodes that connect the families have above 75% bootstrap sup- ionic amino acids and this family still contains three orphan port (see Supplementary data 3). Many of these members are transporters (see Supplementary Table 1) while the SLC12 known to be important in the brain [25] such as the vesicular family has two orphan transporters and the other have Na+, inhibitory amino acid transporter (VIAAT, SLC32A1) and K+ and ClÀ as substrates. many of the SLC38 transporters. We recently identified five The d-group contains the SLC8 and SLC24 families, which new members in the family (SLC38A7-11) [26] and all these contain transporters for Ca2+ and K+. This topology is based place basal in the SLC38 family with relatively long branches. on an alignment with 935 amino acids of which 35% are infor- R. Fredriksson et al. / FEBS Letters 582 (2008) 3811–3816 3813

Fig. 1. Schematic representation of the four major phylogenetic clusters, the a-, b-, c- and d-groups, of SLCs identiﬁed in this study. Each subfamily is color coded, with schematic images describing the predicted structure of the proteins from the respective families. The common name for the diﬀerent families is noted next to the SLC symbol. Phylogenetic trees for all SLC families with at least three members can be found in Supplementary data 3.

mative and the two nodes are joined by a node with a boot- We also calculated phylogenetic trees for all SLC families strap support of 998 (Supplementary data 3). Two transport- with more than three members separately and we present these ers, SLC24A5 and SLC24A6, are orphans and we identified in Supplementary data 3. Six SLC families, containing in total no new members from this family. 10 sequences did not place in the a – d groups according to our 3814 R. Fredriksson et al. / FEBS Letters 582 (2008) 3811–3816 criteria and had fewer than three members and these were ex- symbol (if available) for these until the nomenclature is re- cluded from all phylogenetic calculations. In addition, nine solved. We were able to cluster 15 sub-families (including very divergent sequences could not be placed with certainty SV2) into four main groups, whose alignments had a large into any SLC family and these were also excluded from the number of parsimonious informative sites. It is likely that phylogenetic calculations. We present a summary of the phylo- the sequences we clustered in each of the four main groups genetic analysis from the 46 families in Table 1 and when sum- have a common descent. Interestingly, the eight sub-families marized the human genome contains 384 SLC genes. in the a-group have common features in form of 12 putative We classified each SLC sequence according to (1), the type TM regions, N- and C-termini at the cytosolic side, a large ex- of substrate it transports and (2) the number of transmem- tra cellular loop between TM1 and TM2 (except SLC17) and a brane (TM) regions. The number of TM regions is based on large third intracellular loop. This could be valuable for find- the literature data according to Supplementary Table 1 (Pub- ing the substrate and the functions of the 42 orphan transport- Med ID (PMID) column) or on ab initio predictions, if no ers in this group. The a-group contains an additional six genes information was available, which should obviously not be con- that do not place into any of the known SLC families in the sidered to be a defined fact. The data are presented in Supple- phylogeny. Of these, two have previously been suggested to be- mentary Table 1 and summarized in Fig. 2.InFig. 2a, we long to the SLC22 family [23,24] and one has been assigned to identify 10 major classes of substrates that are being trans- the SLC37 family (Fig. 1). ported by SLCs and we also define classes for orphans (sub- We also found a clear evolutionary relationship between the strate is unknown) and other (substrate that does not fit into proteins from the SLC36, SLC32 and SLC38 families forming any of the 10 classes). In Fig. 2b we display the number of the b-group. The SLC32 are vesicular while the SLC36 and TM regions that the transporters contain, according to evi- SLC38 families are, as far as it is known, situated in the plasma dence from the literature. Fig. 2c also displays the number of membrane [27]. Sequences in the b-group share functional fea- TM regions but based on ab initio predictions with the Phobi- tures as all these transport amino acids, they are all postulated us program (http://phobius.cgb.ki.se/). to have eleven TM regions and they have relatively long N-termini. The N-termini is found at the cytosolic side for SLC32, the vesicular lumen side for SLC36 and at the extracellular side 3. Discussion for SLC38 [27]. In addition SLC7 and SLC12 form a phylogenetic cluster (the c-group) as well as the SLC8 and SLC24 (the This is the first presentation of the gene repertoire and the d-group). first systematic phylogenetic analysis of the hierarchy between The classical definition of the SLCs is that it is a transporter the groups of SLCs in the human genome. We classified 384 that is not driven by ATP [5]. This is obviously a vague defini- human sequences as SLCs. Several of these were not previ- tion in many aspects and there are several sequences that are ously described as SLCs and 19 of these were assigned SLC borderline SLCs from sequence similarity measures. In total names by the HGNC. Yet others could not be placed with cer- nine of the genes discussed in this work code for proteins that tainty into any SLC family, although sequence analysis are clearly most similar to the SLC family, considering our strongly suggests that they do belong to the SLC superfamily. comparison to the entire RefSeq dataset, but these cannot be We chose to use the accession number or some non-SLC gene placed with confidence into any of the phylogenetic clusters.

Fig. 2. Pie charts showing the human SLCs classification. (a) Based on the type of substrate they are transporting. The transporters were classified into ten major groups based on the substrate they are transporting according to the literature. Orphan transporter (substrate unknown) and other (substrate does not fit into any of the ten major categories) are also included as groups in the graph. The distinction between the categories is not always unambiguous and presentation here is somewhat simplified. For example which transporters transport neurotransmitters and amino acids is not completely unambiguous and here we used the distinction that if an amino acid or amino acid derivative is known to function as a neurotransmitter it was placed into the neurotransmitter category, otherwise it was placed in amino acid group. Supplementary Table 1 shows how each single SLC was classified. (b) Based on the number of transmembrane regions according to the literature data. The reference from which this data is obtained is listed in Supplementary Table 1. (c) Based on the number of transmembrane regions the SLCs have in an ab initio prediction performed with the Phobius (http://phobius.cgb.ki.se/) program. The number of transmembrane regions according to Phobius is shown in Supplementary Table 1. R. Fredriksson et al. / FEBS Letters 582 (2008) 3811–3816 3815

Some of these were assigned names from the MFSD# nomen- were aligned using the T-Coffee [29] version 5.72 with default settings. clature (Major Facilitator Superfamily Domain) by the The default alignment parameters were applied. Phylogenetic trees HGNC to indicate their similarity to the SLC family while were calculated as previously described [18]. Briefly, the alignment was bootstrapped 1000 and Maximum Parsimony and Neighbor Join- other have retained their original names, most often derived ing Trees were calculated on these alignments. The topology from the from annotations in large scale cDNA sequencing projects Maximum Parsimony tree was used to calculate Maximum Likelihood for example HIAT (Hippocampus Abundant Transcript). Branch lengths for Fig. 1. Alignment statistics was obtained using Close to 40% (151) of all SLCs are still orphans, and many MEGA 3.1 [30]. of these were just recently described. The largest class of sub- Acknowledgments: We thank Elspeth A. Bruford at the HUGO strates is inorganic ions, with in total 58 SLCs, shown in nomenclature committee for the valuable help on the nomenclature Fig. 2a. This class constitutes mainly metal ion transporters of the genes. R.F. was supported by the Go¨ran Gustafsson Founda- from nine families (SLC5, 9, 11, 12, 20, 24, 30, 39 and 41 fam- tion. These studies were supported by the Swedish Research Council ˚ ilies) but also the sulfate ion transporters from the SLC26 fam- (VR), Svenska La¨karesa¨llskapet, Ahlens Foundation, Lars Hiertas foundation, Gunvor and Josef Aners Foundation, The Royal Swedish ily. In addition, these families include in total 20 orphan Academy of Sciences and Tore Nilsson foundation, the Novo Nordisk transporters, which also are likely to transport some sort of Foundation, and the Magnus Bergvall Foundation. metal ions, which means that the human genome could contain close to 80 transporters for metal ions. The importance of having a regulated transport of amino acids over the plasma and other cellular membranes is clearly Appendix A. Supplementary data shown by the number of genes devoted to this type of function. Taken together there are over 60 known transporters for amino Supplementary data associated with this article can be acids found in seven different SLC families (SLC4, 6, 7, 16, 25, found, in the online version, at doi:10.1016/j.febslet. 36, 38 and 43 for amino acids and SLC17, 1, 6, 18 and 32 for 2008.10.016. neurotransmitters), suggesting that such functions may have arisen multiple times in evolution. In addition, there are 54 or- References phan transporters in these families, and many of those could also be transporters for amino acids. Hence, there could be [1] Yu, F.H. and Catterall, W.A. (2004) The VGL-chanome: a close to 100 SLCs transporting amino acids in the human gen- protein superfamily specialized for electrical signaling and ionic ome which would account for over 25% of the SLC repertoire. homeostasis. Sci. STKE 2004, re15. In summary, we present the first genomic roadmap of the [2] Borst, P. and Elferink, R.O. (2002) Mammalian ABC transporters in health and disease. Annu. Rev. Biochem. 71, 537–592. SLCs. We systematically mined the human genome for SLC [3] Wang, F., Feng, X.C., Li, Y.M., Yang, H. and Ma, T.H. (2006) sequences, and have together with the HGNC provided a suit- Aquaporins as potential drug targets. Acta Pharmacol. Sin. 27, able nomenclature. Moreover, we have analyzed the sequence 395–401. relationship, illustrated the phylogenetic relationships, and [4] Dunbar, L.A. and Caplan, M.J. (2001) Ion pumps in polarized cells: sorting and regulation of the Na+,K+- and H+,K+- found that many of the SLCs can be found in larger groups ATPases. J. Biol. Chem. 276, 29617–29620. that are likely to have common descent. [5] Hediger, M.A., Romero, M.F., Peng, J.B., Rolfs, A., Takanaga, H. and Bruford, E.A. (2004) The ABCs of solute carriers: physiological, pathological and therapeutic implications of hu- 4. Materials and methods man membrane transport proteins Introduction. Pflug. Arch. 447, 465–468. Detailed descriptions of all procedures can be found in Supplemen- [6] Shimokawa, N., Okada, J., Haglund, K., Dikic, I., Koibuchi, N. tary Methods. and Miura, M. (2002) Past-A, a novel proton-associated sugar transporter, regulates glucose homeostasis in the brain. J. Neurosci. 22, 9160–9165. 4.1. Construction of Hidden Markov Models [7] Traiffort, E., Ruat, M., OÕRegan, S. and Meunier, F.M. (2005) We obtained a seeding set of all known 321 SLCs based on the SLC Molecular characterization of the family of choline transporter- database (http://www.bioparadigms.org/slc/menu.asp). The sequences like proteins and their splice variants. J. Neurochem. 92, 1116– from each family were aligned using ClustalW 1.83 [28] which were used 11125. to construct sequence HMMs using the HMMer package. All HMM [8] Shayeghi, M. et al. (2005) Identification of an intestinal heme models are available in HMMer format as Supplementary data 1. These transporter. Cell 122, 789–801. HMMs were used to search the human RefSeq database version 29 [9] Srinivas, S.R., Gopal, E., Zhuang, L., Itagaki, S., Martin, P.M., (ftp://ftp.ncbi.nih.gov/refseq/H_sapiens/), the Ensembl human peptide Fei, Y.J., Ganapathy, V. and Prasad, P.D. (2005) Cloning and database version 29 (ftp://ftp.ensembl.org/pub/current_homo_sapiens/ functional identification of slc5a12 as a sodium-coupled low- data/fasta/pep/), the human MGC cDNA database (ftp://ftp1.nci.nih. affinity transporter for monocarboxylates (SMCT2). Biochem. J. gov/pub/MGC/fasta/) and the Ensembl human ab initio protein data- 392, 655–664. base version 29 (ftp://ftp.ensembl.org/pub/current_homo_sapiens/ [10] Hoglund, P.J., Adzic, D., Scicluna, S.J., Lindblom, J. and data/fasta/pep/). The searches were originally made on RefSeq version Fredriksson, R. (2005) The repertoire of solute carriers of family 29, and Ensembl version 35, and later redone on versions 30 and 36, 6: identification of new human and rodent genes. Biochem. respectively. In the resulting SLC dataset, all duplicate hits, incomplete Biophys. Res. Commun. 336, 175–189. sequences and splice variants were removed as we have previously de- [11] Haitina, T., Lindblom, J., Renstrom, T. and Fredriksson, R. scribed [18]. This resulted in a dataset of 384 sequences. The new se- (2006) Fourteen novel human members of mitochondrial solute quences were grouped with sequences from one of the 46 SLC carrier family 25 (SLC25) widely expressed in the central nervous families based on the highest scoring pair wise alignment. These ex- system. Genomics 88, 779–790. tended SLC families were subjected to phylogenetic analysis. [12] Ishida, N., Kuba, T., Aoki, K., Miyatake, S., Kawakita, M. and Sanai, Y. (2005) Identification and characterization of human 4.2. Phylogenetic analysis Golgi nucleotide sugar transporter SLC35D2, a novel member of Phylogenetic trees were calculated for all the 46 families separately the SLC35 nucleotide sugar transporter family. Genomics 85, using the following procedure. Amino acid sequences in Fasta format 106–116. 3816 R. Fredriksson et al. / FEBS Letters 582 (2008) 3811–3816

[13] Takamori, S. (2006) VGLUTs: exciting times for glutamatergic [22] Portela-Gomes, G.M., Lukinius, A. and Grimelius, L. (2000) research? Neurosci. Res. 55, 343–351. Synaptic vesicle protein 2, a new neuroendocrine cell marker. Am. [14] Visser, W.F., van Roermund, C.W., Waterham, H.R. and J. Pathol. 157, 1299–1309. Wanders, R.J. (2002) Identification of human PMP34 as a [23] Jacobsson, J.A., Haitina, T., Lindblom, J. and Fredriksson, R. peroxisomal ATP transporter. Biochem. Biophys. Res. Commun. (2007) Identification of six putative human transporters with 299, 494–497. structural similarity to the drug transporter SLC22 family. [15] Fredriksson, R., Lagerstrom, M.C., Lundin, L.G. and Schioth, Genomics 90, 595–609. H.B. (2003) The G protein-coupled receptors in the human [24] Gallagher, E., Mc Goldrick, A., Chung, W.Y., Mc Cormack, O., genome form five main families. Phylogenetic analysis, paralogon Harrison, M., Kerin, M., Dervan, P.A. and Mc Cann, A. (2006) groups, and fingerprints. Mol. Pharmacol. 63, 1256–1272. Gain of imprinting of SLC22A18 sense and antisense transcripts [16] Manning, G., Whyte, D.B., Martinez, R., Hunter, T. and in human breast cancer. Genomics 88, 12–17. Sudarsanam, S. (2002) The protein kinase complement of the [25] Mackenzie, B. and Erickson, J.D. (2004) Sodium-coupled neutral human genome. Science 298, 1912–1934. amino acid (System N/A) transporters of the SLC38 gene family. [17] Gloriam, D.E., Fredriksson, R. and Schioth, H.B. (2007) The G Pflug. Arch. 447, 784–795. protein-coupled receptor subset of the rat genome. BMC Genom. [26] Sundberg, B.E. et al. (2008) The evolutionary history and tissue 8, 338. mapping of amino acid transporters belonging to solute carrier [18] Lagerstrom, M.C., Hellstrom, A.R., Gloriam, D.E., Larsson, families SLC32, SLC36, and SLC38. J. Mol. Neurosci.. T.P., Schioth, H.B. and Fredriksson, R. (2006) The G protein- [27] Gasnier, B. (2004) The SLC32 transporter, a key protein for the coupled receptor subset of the chicken genome. PLoS Comput. synaptic release of inhibitory amino acids. Pflug. Arch. 447, 756– Biol. 2, e54. 759. [19] International Human Genome Sequencing Consortium (2004) [28] Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUS- Finishing the euchromatic sequence of the human genome. TAL W: improving the sensitivity of progressive multiple Nature 431, 931–945. sequence alignment through sequence weighting, position-specific [20] Marobbio, C.M., Giannuzzi, G., Paradies, E., Pierri, C.L. and gap penalties and weight matrix choice. Nucleic Acids Res. 22, Palmieri, F. (2008) Alpha-isopropylmalate, a leucine biosynthesis 4673–4680. intermediate in yeast, is transported by the mitochondrial [29] Notredame, C., Higgins, D.G. and Heringa, J. (2000) T-Coffee: a oxaloacetate carrier. J. Biol. Chem.. novel method for fast and accurate multiple sequence alignment. [21] Sawada, K., Echigo, N., Juge, N., Miyaji, T., Otsuka, M., Omote, J. Mol. Biol. 302, 205–217. H., Yamamoto, A. and Moriyama, Y. (2008) Identification of a [30] Kumar, S., Tamura, K., Jakobsen, I.B. and Nei, M. (2001) vesicular nucleotide transporter. Proc. Natl. Acad. Sci. USA 105, MEGA2: molecular evolutionary genetics analysis software. 5683–5686. Bioinformatics 17, 1244–1245.