Báo cáo khoa học: The bile⁄arsenite⁄riboflavin transporter (BART) superfamily docx

REVIEW ARTICLE The bile ⁄ arsenite ⁄ riboflavin transporter (BART) superfamily Nahla M. Mansour*, Mrinalini Sawhney, Dorjee G. Tamang, Christian Vogl and Milton H. Saier Jr Division of Biological Sciences, University of California at San Diego, La Jolla, CA, USA Over the years, our research group has developed a classification system universally applicable to all trans- membrane transporters found in living organisms on Earth [1,2]. This system, adopted by the International Union of Biochemistry and Molecular Biology (IUBMB) in 2003, currently includes about 400 famil- ies of transporters of various types [3]. As a result of the development of sensitive software (gap [4], ic [5]), some of these families have been shown to be distantly related by common descent, and hence they comprise superfamilies [6]. The importance of family and superfamily assign- ment is emphasized by the fact that structural, func- tional and mechanistic data for transporters can be extrapolated from one protein to another if, and only if, they have been shown to be related by common des- cent [1,2]. Further, the degree to which one can extra- polate data from one protein to another is inversely Keywords arsenite; bile acids; cyclic di-GMP metabolism; intragenic duplication; phylogeny; regulation; riboflavin; secondary carriers; topology; transporter Correspondence M. H. Saier Jr, Division of Biological Sciences, University of California at San Diego, La Jolla, CA 92093-0116, USA Fax: +1 858 534 7108 Tel: +1 858 534 4084 E-mail: msaier@ucsd.edu *Present address Vaccines & Recombinant DNA Technology Lab, Nobel Project, NRC, Egypt (Received 21 September 2006, revised 16 November 2006, accepted 4 December 2006) doi:10.1111/j.1742-4658.2006.05627.x Secondary transmembrane transport carriers fall into families and super- families allowing prediction of structure and function. Here we describe hundreds of sequenced homologues that belong to six families within a novel superfamily, the bile ⁄ arsenite ⁄ riboflavin transporter (BART) super- family, of transport systems and putative signalling proteins. Functional data for members of three of these families are available, and they trans- port bile salts and other organic anions, the bile acid:Na + symporter (BASS) family, inorganic anions such as arsenite and antimonite, the arse- nical resistance-3 (Acr3) family, and the riboflavin transporter (RFT) fam- ily. The first two of these families, as well as one more family with no functionally characterized members, exhibit a probable 10 transmembrane spanner (TMS) topology that arose from a tandemly duplicated 5 TMS unit. Members of the RFT family have a 5 TMS topology, and are homol- ogous to each of the repeat units in the 10 TMS proteins. The other two families [sensor histidine kinase (SHK) and kinase ⁄ phosphatase ⁄ synthe- tase ⁄ hydrolase (KPSH)] have a single 5 TMS unit preceded by an N-ter- minal TMS and followed by a hydrophilic sensor histidine kinase domain (the SHK family) or catalytic domains resembling sensor kinase, phospha- tase, cyclic di-GMP synthetase and cyclic di-GMP hydrolase catalytic domains, as well as various noncatalytic domains (the KPSH family). Because functional data are not available for members of the SHK and KPSH families, it is not known if the transporter domains retain transport activity or have evolved exclusive functions in molecular reception and sig- nal transmission. This report presents characteristics of a unique protein superfamily and provides guides for future studies concerning structural, functional and mechanistic properties of its constituent members. Abbreviations aas, amino acyl residues; Acr3, arsenical resistance-3; BASS, bile acid:Na + symporter; BART, bile ⁄ arsenite ⁄ riboflavin transporter; HATPase-c, histidine kinase-like ATPase; KPSH, kinase ⁄ phosphatase ⁄ synthetase ⁄ hydrolase; RFT, riboflavin transporter; SD, standard deviations; SHK, sensor histidine kinase; TCDB, Transporter Classification Database; TMS, transmembrane spanner; UNK, unknown. 612 FEBS Journal 274 (2007) 612–629 ª 2007 The Authors Journal compilation ª 2007 FEBS related to their phylogenetic distances [7–11]. Import- antly, bioinformatic procedures can reveal the evolu- tionary pathways taken for the appearance of the proteins [12,13]. The criterion we have been using for the establish- ment of homology is a comparison score of nine stand- ard deviations (SD) or greater using the gap and ic programs. These programs correct for unusual or restricted amino acyl residue compositions as occur in integral membrane proteins. The value of nine SD cor- responds to a probability of 10 )19 that the degree of sequence similarity observed could have occurred by chance [1,14]. This is a highly reliable criterion of homology that is far more rigorous than most other criteria currently in use by the scientific community. One recently identified superfamily was shown to include the bile acid:Na + symporter (BASS) family (TC #2.A.28) and the arsenical resistance-3 (Acr3) family (TC #2.A.59) [6]. However, except for a brief, outdated description of the BASS family in 1999 [15] and the establishment of a common origin for these two families [6], the characterization of this small superfamily had not been reported previously. We have conducted sequence comparisons which revealed that the recently characterized riboflavin transporter (RFT) of Lactococcus lactis, RibU [16] is a member of a moderately sized family of five putative a-helical transmembrane spanning (TMS) proteins that is distantly related to the 10 TMS transporters of the BASS and Acr3 families. This first became apparent following BLAST searches of the Transporter Classifi- cation Database (TCDB). In this paper, we show that the putative 10 TMS proteins of the latter two families arose by intragenic duplication of an element encoding a 5 TMS protein similar to RibU and its orthologue in Bacillus subtilis , YpaA. We further identify addi- tional families within this ubiquitous superfamily, demonstrating the presence of six families, three with a single 5 TMS repeat unit and three with two, dupli- cated, 5 TMS repeat units. Most unexpectedly, two of the three families with a single 5 TMS unit exhibit fea- tures of catalytic proteins. One of these families, the SHK family, is a coherent group of proteins of similar structure with the N-terminal hydrophobic transporter domain linked to a C-terminal hydrophilic sensor his- tidine kinase (SHK) domain. The other, the KPSH family, is a heterogeneous group of multidomain pro- teins, each exhibiting a different set of domain combi- nations, suggesting differing catalytic and regulatory functions. Catalytic domains in these proteins include kinases, phosphatases, cyclic di-GMP synthetases and cyclic di-GMP hydrolases (KPSH). None of the four members of the KPSH family have been functionally characterized, but the sequence similarity with charac- terized proteins and protein domains allows us to make functional predictions with a high degree of con- fidence. The SHK and KPSH families have been briefly described previously [17]; they are listed in the Pfam and Interpro databases as the ‘5 TM receptors of the 5TMR-LYT domain’ (PF07694) and the ‘5 TM receptors of the LytS-YhcK type transmembrane region’ (IPR011620), respectively. Finally, one of the families (UNK or unknown family), consisting of putative transporters with two tandemly repeated 5 TMS units, includes homologues with no function- ally characterized members. In this case, we have no basis for making confident predictions of substrates transported or the energy coupling mechanism(s) involved. The observations reported here reveal that this superfamily is far more diverse than was previ- ously recognized. Results Homologues with a basic 5 TMS unit Using the 5 TMS riboflavin transporter (YpaA; Bsu1 in Table S1) of B. subtilis as the query sequence, PSI- BLAST searches against the NCBI protein database with iterations [18] brought up the homologues listed in Tables S1–S3 on our website (http://biology.ucsd. edu/msaier/supmat/BART/). These include the char- acterized riboflavin transporter, RibU of L. lactis (Lla3 in supplementary Table S1) [16]. The what pro- gram [19] was used to predict the topologies of individ- ual proteins. A multiple alignment was derived using the clustal x program [20], and the treeview pro- gram [21] was used to draw a phylogenetic tree (data not shown). This tree revealed that these proteins fall into three subfamilies which we call the RFT (58 proteins), SHK (31 proteins) and KPSH (4 proteins) families (see above). While the RFT family includes both bacterial and archaeal proteins, the SHK and KPSH families included members that are derived exclusively from bacteria. Most of these bacterial proteins are from either proteobacteria or firmicutes (Tables S1–S3). Using the gap and ic programs [4,5], we established that all of the proteins listed in Tables S1–S6 are homologous in the regions of their transmembrane domains (see below). The hydrophobic domains of members of the three 5 TMS families are relatively similar in sequence to each other, as are those of mem- bers of the three 10 TMS families (comparison scores of ‡ 15 SD). The 5 TMS proteins are more distantly related to the 10 TMS proteins. N. M. Mansour et al. The BART superfamily FEBS Journal 274 (2007) 612–629 ª 2007 The Authors Journal compilation ª 2007 FEBS 613 Homologues with a basic 10 TMS unit To identify the protein homologues of the 10 TMS families of the bile ⁄ arsenite ⁄ riboflavin transporter (BART) superfamily, the ArsB protein of B. subtilis (P45946) was used as the query sequence in PSI- BLAST searches conducted with 11 iterations. Hun- dreds of homologues were retrieved. Redundancies were removed, leaving 285 protein sequences (supple- mentary Tables S4–S6). About 82% are from bac- teria while 15% are from eukaryotes, and 3% are from archaea. A phylogenetic tree was generated (data not shown). The proteins proved to fall into three major families BASS, Acr3 and UNK. All three families contain proteins from bacteria, archaea and eukaryotes, and all three families include pro- teins from both Gram-positive and Gram-negative bacteria. However, there are some organismal distinc- tions. For example, within the eukaryotic domain, the BASS family has homologues from plants, ani- mals and fungi, but the Acr3 family has only fungal protein members, and the UNK family consists only of animal and plant proteins. These distinctions undoubtedly correlate with distinctive functions. The fact that eukaryotes have 10 TMS members of the BART superfamily, but not 5 TMS members, may reflect the tendency of eukaryotic proteins to become larger during evolution, possibly for purposes of complex formation, subcellular targeting and regula- tion [22]. In the archaeal domain, only one archaeal subdivi- sion, the Euryarchaeota, is represented. However, the genuses represented differ depending on the family. The BASS family has homologues only from Pyrococ- cus, the Acr3 family has proteins from Archaeoglobus, Pyrococcus and Thermococcus, and the UNK family has homologues only from Haloarcula and Methano- sarcina. The low representation of archaeal homo- logues is worthy of note. The BASS and UNK families have equal numbers of eukaryotic homologues (23 and 24, respectively), about 23% and 16%, respectively, of the total numbers of members of these two families. The Acr3 family has just 5% of its members derived from eukaryotes. Many organisms encode within their genomes more than one paralogue of the 10 TMS BART superfamily proteins, but few if any seem to encode more than four. No archaeon has more than one. Among the eukaryotes, the fungi appear to have just one or two per organism while most fully sequenced genomes of plants and animals encode either two or three. Bacteria represented have one to four 10 TMS para- logues. Preliminary evidence for homology of the 5 and 10 TMS proteins of the BART superfamily When TCDB was blasted using TC-BLAST [18,25] with the YpaA protein of B. subtilis (Bsu1 in cluster 3 of Fig. 1B) as the query sequence, the ArsB protein of B. subtilis was retrieved with an e-value of 0.006. Resi- dues 22–187 in YpaA (TMSs 2–5) aligned with resi- dues 25–167 in ArsB (TMSs 2–5) showing 26% identity and 42% similarity. When the best conserved region of this binary alignment was examined with the gap program and 500 random shuffles, a comparison score of 7.0 SD was obtained. These values are already sufficient to suggest, but not establish, homology. The sequence similarity between the 5 TMS proteins and the first repeat unit of the 10 TMS proteins was substantially greater than observed when the 5 TMS protein sequences were compared with the second repeat units of the 10 TMS proteins. This observation led us to suggest that when the 5 TMS proteins (which presumably function as homodimers) duplicated to give 10 TMS proteins, the first repeat unit retained its original topology and its primary, generalized, trans- port function, while the second repeat unit diverged in sequence to a greater extent to assume the opposite topology in the membrane and to serve a more special- ized, permease-specific function. A generalized function might, for example, be energy coupling, while a specialized function might be substrate recognition. Precedence for these concepts has been published pre- viously [26–30]. Homology between the 5 TMS and 10 TMS proteins is established below. The riboflavin transporter (RFT) family The proteins of the RFT family within the BART superfamily are presented in Table S1, and the multiple alignments of their sequences are shown in Figure S1A on our website. In Table S1 and sub- sequent tables, the proteins are arranged first accord- ing to phylogenetic cluster, and second according to position in that cluster. Using the what program [19] and the hmmtop program [31], most homologues have five putative TMSs although some were predicted to have six (Table S1). The average hydropathy and similarity plots for this family are shown in Fig. 1A. There are five peaks of average hydrophobicity corresponding to five peaks of average similarity. It therefore appears that these pro- teins share a common 5 TMS topology. The amphi- pathicity plot (not shown) revealed no distinctive characteristics. The multiple alignments upon which these plots were based (Fig. S1A) showed no single The BART superfamily N. M. Mansour et al. 614 FEBS Journal 274 (2007) 612–629 ª 2007 The Authors Journal compilation ª 2007 FEBS residue position with full residue conservation or even full conservation of residue type. However, as shown in Fig. 1A, TMS 2 is best conserved. It shows the fol- lowing consensus sequence: D FSDVPðHyÞ 3 G G ðHyÞ 3 GPðHyÞ 2 G ðHyÞ 6 KNðHyÞ 3 Y ðHyÞ 2 XGX 3 G (alignment positions 76–114; X, any residue; Hy, any hydrophobic residue; italic residues, consensus residues that are common to those in the SHK family; under- lined residues, consensus residues that are common to those in the KPSH family.) The clustal x-derived phylogenetic tree of the RFT family is shown in Fig. 1B, and the bootstrapped tree is shown in Figure S1B. We also derived paup-based trees using both neighbor joining and parsimony algorithms (Figs S1C and S1D, respectively). Neighbor joining bootstrapped trees for all six families (supplementary Figs S1–6B and S1–6C) as well as parsimony trees (sup- plementary Figs S1–6D) are provided on our website. The neighbor joining and parsimony trees, with or Va lu e Alignment Position -0.5 0 0.5 1 1 100 200 A B 12 3 4 5 -1 Tma1 Tko1 Pab1 Pho1 Cac3 Cac2 Cte2 Sth3 Rxy1 Bac1 Tte3 Lca2 Lde2 Lac2 Lga2Ljo2 Lpl2 Ppe2 Efa4 Efa5 Lla4 Sth7 Spy3 Ssu2 Spn4 Sag4 Smu3 Lla3 Ssu1 Smu1 Spy2 Sag1 Spn2 Sth2 Lme1 Ooe2 Lmo3 Lin1 Gka1 Bsu1 Bli1 Bce1 Ban1 Bth1 Oih1 Sau2 Sep1 Cpe1 Tte1 Cac1 Cte1 Blo2 Lpl1 Ppe1 Efa1 Lca1 Lga1 Ljo1 1 2 3 4 Fig. 1. (A) Average hydropathy (top) and similarity (bottom) plots for the RFT family. The AVEHAS program [79] was used to gener- ate the plots shown here (and elsewhere in this paper), based on the CLUSTAL X [20] mul- tiple alignment as shown in Fig. S1. The proteins and their properties are tabulated in Table S1A on our website (http://biology. ucsd.edu/msaier/supmat/BART). (B) Phylo- genetic tree of the RFT family. The tree is based on the CLUSTAL X alignment shown in Fig. S1A. The bootstrapped tree is shown in Fig. S1B. PAUP-based trees [76] based on neighbor joining (Fig. S1C) and parsimony (Fig. S1D) are also available on our website. All tables of the proteins (Tables S1–S6), multiple alignments of the protein members of the six families of the BART superfamily (RFT, SHK, KPSH, BASS, ACR and UNK; Figs S1A–S6A) as well as the bootstrapped trees (Figs S1B–S6B) can be found on our website (http://biology.ucsd.edu/msaier/ supmat/BART). PAUP trees designed using neighbor joining (with bootstrapping) and parsimony (without bootstrapping) can be found on our website in Figs S1C–S6C, and S1D–S6D, respectively. The format of pres- entation is the same for Figs 2–6. N. M. Mansour et al. The BART superfamily FEBS Journal 274 (2007) 612–629 ª 2007 The Authors Journal compilation ª 2007 FEBS 615 without bootstrapping, are very similar. Four clusters are apparent. Cluster 1 consists of proteins from firmi- cutes and one Actinobacterium, Rubrobacter xylanophi- lus (Rxy1). This last protein falls into a subcluster with three firmicute proteins, showing that although most of these proteins follow the phylogenies of the host organ- isms, this is not true of Rxy1. Proteins in cluster 1 show a broader size range [176–222 amino acyl residues (aas)] than for the other three clusters, but proteins in all four clusters are of similar sizes. Cluster 2 proteins are exclusively from firmicutes, and seven of the nine homologues have paralogues in cluster 1. The other two (Lme1 and Ooe2) apparently lack paralogues in the RFT family. All but two pro- teins in cluster 3 are from firmicutes, and the two exceptions (Tma1 and Blo2) are distantly related to each other and all other homologues of cluster 2. Clus- ter 2 contains the characterized riboflavin transporter, RibU, from Lactococcus lactis (Lla3) [16]. Cluster 3 contains the functionally characterized ribo- flavin transporter of B. subtilis (Bsu1; YpaA; C. Vogl, unpublished results). Because there is extensive overlap of organismal sources between clusters 1 and 2, as well as between clusters 1 and 3 (but not between clusters 2 and 3), we suggest that the proteins in cluster 1 primar- ily represent one set of functionally related orthologues, different from those in clusters 2 and 3, which may, however, all be orthologous, serving a single function. Three archaeal proteins comprise cluster 4. These proteins are also likely to be orthologous to each other, possibly also to cluster 1 or cluster 2 ⁄ 3 proteins. The SHK family Thirty-one proteins comprise the current SHK family (supplementary Table S2 and Figs S2A–D). These pro- teins have an N-terminal 6 TMS hydrophobic domain (Fig. 2A) where TMS 0 is unique to the SHK family. It is, however, well conserved, suggesting that it serves an important unified function in proteins of the SHK family. TMSs 1–5 in Fig. 2A correspond in sequence to TMSs 1–5 in the RFT family (Fig. 1A). Note that in both Figs 1A and 2A, TMSs 1, 2 and 4 are more hydrophobic than peaks 3 and 5. The 6 TMS hydrophobic domain in SHK family proteins is followed by three recognizable domains. The first is a cGMP-binding phosphodiesterase ⁄ Anabaena adenyl cyclase ⁄ E. coli FhlA (GAF) domain, present in phytochromes, cyclic GMP phosphodiest- erases and other sensory transduction proteins [32]. The second is a large, well conserved sensor kinase domain, homologous to thousands of other sensor kin- ases in the NCBI database. Those included in this study are all more similar to each other than they are to any of the other sensor kinases, and only these have the homologous N-terminal hydrophobic domain com- mon to the RFT family proteins. The third domain is the HATPase-c domain, a histidine kinase-like ATPase domain. These domains are found not only in sensor kinases, they are also found in topoisomerases I and II, heat shock proteins of the HSP90 family, phytochrome ATPases and DNA mismatch repair enzymes [33]. Because sensor kinase domains must be in the cyto- plasm, we can infer that TMS 0 (Fig. 2A) passes through the membrane from inside the cell to the outside. By analogy, the 5 TMS proteins of the RFT family may have their N-termini outside and their C-termini inside (see below). Examination of the SHK family multiple alignments (Fig. S2A) revealed many fully conserved residues. The most condensed region of conservation within the hydrophobic domain occurred in TMS 2 where the consensus sequence is: N T R ðHy Þ 2 G ðHyÞ 3 ðGÞ G ðHyÞ 2 G G P ðHyÞ 2 G ðHyÞ 3 G LTGG L HRYSHyG (alignment positions 113–145; Hy, any hydrophobic residue; bold, fully conserved; italic residues, common to the consensus sequence residues for the RFT family; underlined residues, common to those of the KPSH family). A few fully conserved positions are also present in TMSs 3, 4 and 5 as well as the downstream hydro- philic domains. The latter include an A VAI T DREKI L A consensus region with three fully con- served residues (alignment positions 292–303). Exam- ination of Table S2 and Fig. 2B reveal that only one organism, Photobacterium profundum, has more than one SHK member, and the two paralogues in this organism are distantly related, falling into different clusters of the phylogenetic tree. With two exceptions, proteins of the SHK family are of fairly uniform size (556–597). Both firmicutes and proteobacteria as well as one homologue from Fusobacterium nucleatum, are represented. All members of the SHK family are pre- dicted to have six TMSs (Table S2). The phylogenetic tree for the SHK family (Fig. 2B) shows four clusters. Bootstrap values are provided in Figures S2B and S2C. Cluster 1 proteins are exclu- sively from firmicutes, while cluster 3 and 4 proteins are exclusively from proteobacteria. Each of these three clusters is coherent with all proteins within any one cluster branching from each other at points distant from the center of the tree. The two short variants, Ahy1 and Ppr2 (440 and 451 aas), from Aeromonas The BART superfamily N. M. Mansour et al. 616 FEBS Journal 274 (2007) 612–629 ª 2007 The Authors Journal compilation ª 2007 FEBS hydrophila and Photobacterium profundum, respectively, comprise cluster 3. Cluster 2 is most diverse in sequence as well as organismal source. These loosely clustered proteins are from proteobacteria, firmicutes and Fusobacterium nucleatum. The presence of this cluster clearly suggests that members of the SHK fam- ily are not all orthologous. The KPSH family Four proteins comprise the KPSH family (Table S3). These proteins are about equally diverse in sequence as revealed by the phylogenetic tree shown in Fig. 3B. Each is from a different bacterial subdivision, one from Deinococcus geothermalis, one from a c-proteo- bacterium, one from a d-proteobacterium, and one from a putative uncultured archaeon. The tree is based on the multiple alignment shown in Fig. S3A. These proteins exhibit six N-terminal peaks of hydrophobici- ty (peaks 0–5 in Fig. 3A), corresponding to TMSs 0–5 in Fig. 2A. TMSs 1–5 correspond to TMSs 1–5 in Fig. 1A. As with TMS 2 of the RFT and SHK famil- ies, TMS 2 of the KPSH family is the best conserved with the following consensus sequence: G ðHyÞ 3 D Hy R X ðHyÞ 5 X G LFXG XLPðHyÞ 10 YRLXHyG G (alignment positions 72–110 in Fig. S3A; X, any resi- due; Hy, any hydrophobic residue; bold, fully con- served; italic residues are common to those in the RFT family consensus sequence; underlined residues are -1.5 -1 -0.5 0 0.5 1 1 100 200 300 400 500 600 A B 0 1 2 3 4 5 Ban2 Bsu2 Bli2 Ppe3 Efa3 Ooe1 Sag3 Smu2Dde1 Dvu1 Bcl1 Tte2 Fnu1 Ppr2 Ahy1 Dac1 Son1 Ppr1 Vfi1 Vch1 Vvu1 Vpa1 Eam1 Sen1 Sty1 Sfl1 Eco1 Rru1 Eca1 Cvi1 Dar1 12 3 4 Fig. 2. (A) Average hydropathy (top) and similarity (bottom) plots for the SHK family. (B) Phylogenetic tree for the SHK family. The multiple alignment and list of proteins used are presented in Fig. S2A and Table S2, respectively. Four homologues of abnormal size, listed in Table S2, were eli- minated when the Fig. S2A alignment was derived. The bootstrapped trees are shown in Figs S2B and S2C. The parsimony tree is shown in Fig. S2D. N. M. Mansour et al. The BART superfamily FEBS Journal 274 (2007) 612–629 ª 2007 The Authors Journal compilation ª 2007 FEBS 617 common to residues in the SHK family consensus sequence.) The four proteins that comprise the sequence diver- gent members of the KPSH family are all multidomain proteins that seem to share only the characteristic of having a common N-terminal hydrophobic domain. In the case of Son2 from Shewanella oneidensis (998 resi- dues), following the N-terminal 6 TMS domain are three PAS helix–loop–helix, protein–protein interaction domains, common in proteins involved in energy sens- ing and signal transduction [34,35], a GGDEF domain (domain containing the conserved GDEF motif) and an EAL domain (domain containing the conserved EAL motif). The latter two domains are likely to be involved in cyclic di-GMP synthesis and hydrolysis, respectively [36–38]. The Uar2 protein, from an ‘uncultured archaeon’ is of 654 aas and has (following the common N-terminal hydrophobic domain) a LytS domain followed by a COG4191 domain (of unknown function), a histidine kinase A dimerization phosphoacceptor (HisKA) domain, and a C-terminal HATPase-c domain. The LytS domain is homologous to LytS, a signal transduc- tion regulator of cell autolysis [17]. The HisKA domain is a conserved bacterial histidine sensor kinase domain [39], and the HATPase-c domain resembles a histidine kinase ATPase domain [40]. Uar2 is similar in several of these respects to members of the SHK family. Following the N-terminal 6 TMS domain of the Gsu1 protein from Geobacter sulfurreducens are at least two AtoS-type sensor kinase domains [41], followed by (a) a HisKA domain, (b) a HATPase-c domain, and (c) a signal receiver (REC) domain at the extreme C-termi- nus of this 1112 residue protein. Finally, the Dge1 pro- tein from Deinococcus geothermalis is relatively short (349 residues) with a single GGDEF domain following the hydrophobic transmembrane domain. The BASS family Functionally characterized members of the BASS fam- ily catalyze Na + :bile acid symport [15,42]. These sym- porters exhibit broad specificity, taking up a variety of nonbile organic compounds as well as taurocholate and other bile salts [43]. They have been identified in intestinal, liver and kidney tissues of animals, and at least three isoforms are present in a single species such as humans. The BASS family is also called the solute carrier family 10 [23,24,43]. Functionally characterized members of the BASS family appear to possess their bile acid binding sites within and preceding the last transmembrane spanner [23,44]. A BASS in the apical membrane of the human ileal intestine catalyzes the electrogenic uptake of bile acids with a stoichiometry of bile acid:Na + of 1 : 2 [24]. This protein is associated with the 16 kDa subunit c of the vacuolar proton pump, an association that may in part account for its apical location [45]. Thus, the vacuolar proton pump-associated apical sorting machinery may play a role in sorting the apical Na + :bile symporter to the basolateral membrane. The rat liver Na + ⁄ taurocholate cotransporter is sub- ject to elaborate regulation in response to cyclic AMP and cell swelling [46,47]. It has two N-terminal, B -1.5 -0.5 0.5 Value Alignment Position -1 0 1 1 100 200 300 400 500 600 700 800 900 1000 1100 A 0 1 2 3 4 5 Gsu1 Uar2 Dge1 Son2 Fig. 3. (A) Average hydropathy (top) and similarity (bottom) plots for the four proteins of the KPSH family. (B) The phylogenetic tree for these four proteins. The multiple alignment (Fig. S3) and list of pro- teins (Table S3) are available on our website. The bootstrapped trees are shown in Figs S3B and S3C. The parsimony tree is shown in Fig. S3D. The BART superfamily N. M. Mansour et al. 618 FEBS Journal 274 (2007) 612–629 ª 2007 The Authors Journal compilation ª 2007 FEBS N-linked carbohydrate sites and two Tyr-based basolat- eral sorting motifs at its carboxyl terminus (YEKI and YKAA). The former targets the protein to the apical membrane in the absence of the latter, but the latter overrides the former, targeting the protein to the baso- lateral membrane [48]. The ileal homologue has a 14-residue cytoplasmic tail with a b-turn structure that targets the protein to the apical membrane [49]. The human orthologue of the rat Na + ⁄ taurocholate symporter (TC #2.A.28.1.1) (NTCP; SLC10A1) exhib- its multiple single nucleotide polymorphisms in popula- tions of European, African, Chinese and Hispanic people [44]. Four nonsynonymous single nucleotide polymorphisms are associated with significant loss of transport function or change in substrate specificity. One form, found in Chinese Americans, does not cata- lyze bile acid uptake but catalyzes estrone sulfate uptake. This transporter may play a role in mainten- ance of enterohepatic recirculation of bile acids [44]. The members of the BASS family can be found on our website (Table S4), and the clustal x alignment of their sequences, shown in Fig. S4A, provides the basis for the average hydropathy and similarity (Ave- HAS) plots shown in Fig. 4A as well as the tree shown in Fig. 4B and the bootstrapped trees shown in Figs S4B and S4C. As revealed in Table S4, most organisms represented have only one member of the BASS family, but two can be found in a few bacteria, plants and animals, and animals can have up to three. Only Bos taurus and Tetraodon nigroviridis have three. Most of the homologues from prokaryotes fall into the size range 300–350 aas although a few are smaller or larger. The plant proteins are about 400 aas in length, and the animal homologues range from about 350–550 aas with one protein from the chicken having 679 aas. The average hydropathy and similarity plots reveal 10 conserved peaks of average hydropathy. Striking peaks of amphipathicity were observed just preceding peak 1, between peaks 2 and 3, and between peaks 4 and 5, although striking peaks of average amphipathic- ity were not observed in the second hydrophobic halves of these proteins (data not shown). Only the chicken homologue, Gga1, has an extension following peak 10, and only Gga4 has an internal deletion not found in the other homologues. These could be due to errors in exon recognition. The Tni1 protein from Tetraodon nigroviridis has several internal hydrophilic insertions. Several proteins have N-terminal hydrophi- lic extensions, but Gga4 has the longest. A single resi- due proved to be fully conserved in all members of the BASS family. This is a prolyl residue at alignment position 451 in TMS 5. The best conserved regions overlap the moderately hydrophobic peaks 4 (best conserved) and 9 (less well conserved). The consensus sequences for these two peaks are: TMS 4: Hy A V G ðHyÞ 4 GCCPGGTASN ðHyÞ 2 ðSTÞ FLALGDV TMS 9: R ðSTÞ Hy ðSTGÞ FHyGHyQNðSTGÞ GLðAGCÞðHyÞ 4 (Hy, any hydrophobic residue; residues in parentheses represent alternative possibilities at a single position.) The BASS family trees are shown in Figs 4B, and Supplementary figures S4B, S4C and S4D, all of which show excellent agreement as usual. The trees show eight primary clusters as well as several branches that stem from the center of the tree and therefore do not belong to one of the primary clusters. Each of these branches bears a bacterial protein. This tree reveals that BASS family members cluster primarily according to organismal type (also Table S4). Thus, clusters 1–2 consist only of prokaryotic proteins, including both bacterial and archaeal proteins; the small cluster 3 pro- teins are derived only from proteobacteria; cluster 4 proteins are from plants and cyanobacteria; cluster 5 proteins are from a range of nonproteobacterial types; cluster 6 and 7 proteins derive exclusively from ani- mals; and cluster 8 is derived only from bacteria. Although bacterial paralogues were not observed in clusters 1–4, the proteins in none of these clusters fol- lowed the phylogenies of the host organisms. Perhaps early extragenic duplication events followed by nonse- lective gene loss or horizontal transfer of the encoding genes account for these results. Only rice, with two paralogues in cluster 4 (Osa2 and Osa3) has more than one homologue in any one of these clusters. In contrast to clusters 1–4, the clustering patterns in cluster 5 follow those of the source organisms. Because each protein is derived from a different organism, these proteins may be orthologues serving a single function. Like clusters 1–4, the animal proteins in clusters 6 and 7 and the bacterial proteins in cluster 8 are not likely to be orthologous although subclusters of potential orthologues can be identified. For example, the cluster- ing of a spirochete protein (Lin5) with the cyanobacte- rial homologues is unexpected, and possibly resulted from horizontal gene transfer between subdivisions. The ACR3 family Two proteins of the Acr3 family have been function- ally characterized. These proteins are the ‘Acr3’ pro- tein of Saccharomyces cerevisiae, also called the Arr3 protein [50], and the ‘ArsB’ protein of Bacillus subtilis [51]. The latter protein is not related to ArsB of N. M. Mansour et al. The BART superfamily FEBS Journal 274 (2007) 612–629 ª 2007 The Authors Journal compilation ª 2007 FEBS 619 Escherichia coli. The Acr3 protein is present in the yeast plasma membrane and pumps arsenite, but not arsenate, antimonite, tellurite, cadmium or phenyl- arsine oxide out of the cell in response to the proton motive force [50]. The Bacillus protein exports both arsenite and antimonite [51]. The exact transport mechanism is not established, but a uniport or cation antiport mechanism seems probable. Table S5 and Fig. S5A on our website present the members of the Acr3 family and show the clustal x multiple alignment, respectively, upon which the aver- age hydropathy (Fig. 5A, top) and average similarity (Fig. 5A, bottom) plots as well as the phylogenetic tree (Fig. 5B) are based. The bootstrapped and parsimony trees are shown in Figs S5B, S5C and S5D on our website. Examination of Table S5 reveals that most organisms represented have only one Acr3 homologue, and those with two are all from bacteria. No archaeon or eukaryote displays more than one, and no organ- isms had more than two. Examination of the size variations observed for these proteins revealed that most of the prokaryotic Alignment Position Value -0.5 -1.5 0 0.5 1 1 100 200 300 400 600 700 800 900 1 2 3 4 5 6 7 8 9 10 1500 -1 1 A B Aae1 Kra2 Asp4 Bli4 Son2 Wsu1 Dac2 Csa1 Mma7 Mac2 Gox2 Bfu3 Sen4 Sty2 Cef2 Cdi1 Mma5 Asp3 Bpa1 Sco2 Pae3 Psy Pfl1 Sau3 Msu2 Nme3 Oih1 Bli3 Bsu2 Bcl1Bha1 Ban2 Bth5 Lme1 Sth1 Smu1 Nme4 Msu1 Hso1 Osa2 Les1 Ath1 Osa3 Mca2 Sel4 Ssp6 Ftu1 Spo1 Ava3 Sav2 Bli5 Gka3 Bha2 Bcl3 Pgi1 Bfr1 Bth6 Dme3 Cbr1 Jsp1 Mmu7 Bta1 Gga4 Tni1 Bta2 Gga1 Ocu1 Cfa1 Ptr1 Ppy1 Rno1 Cgr1 Dre1 Tni2 Bta3 Tni4 Ppr2 Mde2 Mma9 Lin5 Sel6 Ssp7 Cwa2 Ava2 Nsp2 Pae6 Ppu3 Rge3 Bab1 Bme1 6 7 1 2 3 4 5 8 Hsa5 2 Fig. 4. (A) Average hydropathy (top) and similarity (bottom) plots for the BASS family. (B) The phylogenetic tree for the BASS fam- ily proteins. The list of proteins and the mul- tiple alignment upon which these plots were based can be found in Table S4 and Fig. S4A on our website, respectively. The bootstrapped trees are shown in Figs S4B and S4C. The parsimony tree is shown in Fig. S4D. The BART superfamily N. M. Mansour et al. 620 FEBS Journal 274 (2007) 612–629 ª 2007 The Authors Journal compilation ª 2007 FEBS homologues are of similar sizes (320–390 aas) with just a few exceptions. All of the fungal proteins are larger (389–454 aas), and the two Mycobacterial orthologues are still larger (498 aas). The latter two proteins have hydrophilic C-terminal extensions of about 140 resi- dues. These extensions correspond to the entirety of low molecular weight phosphatases of the LMWP family, some of which (e.g., Wzb of E. coli; P0AAB2; 147 aas) hydrolyze phosphotyrosine proteins, regulating capsular exopolysaccharide production [52–54]. Possibly these transporters play a role in polysaccharide secretion. The fungal homologues proved to have either a  50 residue hydrophilic insertion between putative TMSs 8 and 9, or an N-terminal hydrophilic extension in front of TMS 1, both of unknown function. The average hydropathy and similarity plots reveal 10 well con- served peaks of hydrophobicity (1–10) as well as an additional C-terminal peak (11) present in several homologues, but not in many others. Two prolyl resi- dues are fully conserved, one at alignment position 185 in TMS 3 and the other at alignment position 337 in TMS 6. Nevertheless, the best conserved peaks over- all were TMSs 4 and 9 as for the BASS family. The consensus sequences for these two TMSs are: TMS 4: G A A P C T A A ðHyÞ 3 WSXHyðASTÞ XG ðDETÞ PXðFYÞðTACÞ TMS 9: A A P ðSAÞ 2 ðHyÞ 2 GASNFFEHyAHyA Hy A Hy ðSAGÞ Hy F G (Hy, any hydrophobic residue; residues in parentheses represent alternative possibilities at a single position.) Phylogenetic trees for the Acr3 family are shown in Figs 5B, S5B, S5C and S5D, all in good qualitative agreement. Of the eight bacteria having two para- logues, all but one (Dechloromonas aromatica) have one of these paralogues in cluster 1 and the other in cluster 3. D. aromatica has one in cluster 2 and one in cluster 3. It is interesting to note that bacterial and archaeal proteins are found in all three clusters, but the eukaryotic proteins are all in cluster 3. These fun- gal proteins cluster together, distant from any of the bacterial proteins which cluster into two distinct sub- clusters of cluster 3. The functionally characterized arsenite exporters, Sce1 of Saccharomyces cerevisiae, and Bsu1 of B. subtilis, are in the fungal and pro- karyotic subclusters of cluster 3, respectively (see below). Cluster 1 is diffuse, consisting of distantly related proteins. Subclusters correspond to specific types of bacteria (firmicutes or proteobacteria). The same is observed for some of the subclusters in the more com- pact cluster 2, but there are also some notable exceptions [e.g., Cth1 (from a firmicute) clusters with proteobacterial proteins, and Rpa1 (from a Plancto- mycetes) clusters with Msp1 from an a-proteobacteri- um]. The two primary subclusters in cluster 3 include proteins exclusively from fungi and exclusively from bacteria and archaea, respectively. The latter subclus- ter is split into two subsubclusters, one derived from Actinobacteria with one exception (Mma1 from Mag- netospirillum magnetotacticum,ana-proteobacterium), the other derived from various other prokaryotic sub- divisions, but not from Actinobacteria. This last one includes proteins from proteobacteria, firmicutes, cyanobacteria, chlorobi and euryarchaeota. The UNK family The members of the UNK family are listed in Table S6, and the multiple sequence alignment is shown in Fig. S6A. The latter provided the basis for the average hydropathy and similarity plots shown in Fig. 6A and the tree presented in Fig. 6B. The UNK proteins are derived from eukaryotes (animals, plants and fungi) and bacteria (proteobacteria and actinobac- teria primarily). No two UNK family proteins are derived from a single organism. The average hydropathy plot reveals 10 conserved peaks of hydropathy. A single strong peak of amphi- pathicity (angle set at 100°) was observed between putative TMSs 6 and 7 (data not shown). As expected, based on the properties of the previously described families, peaks 4 and 9 were only weakly hydrophobic. Several fully conserved residues were found: prolyl and glycyl residues in peak 4, a P in peak 6, a K pre- ceding peak 9, and a P and a Q in peak 10. The best conserved peaks were 4 and 5, and 9 and 10 (Fig. 6A). One protein, Mgr2, had an internal deletion near the N-terminus as well as a long C-terminal extension of about 300 residues. Consensus sequences for the four best conserved regions are: P4: G ðHyÞ 4 CX LP ðSTÞTVQS SIAFTSHyAKGNV P9: F C G S K K SLAðSTÞ GHyPMAXHyHyF P5: S S ðHyÞ 2 G ðHyÞ 3 TPðHyÞ 3 TPðHyÞ 3 G ðHyÞ 3 P10: GðHyÞ 4 P ðHyÞ 3 FHQ IQ L MVCAðHyÞ 2 (X, any residue; Hy, any hydrophobic residue; bold, fully conserved.) Limited sequence similarity can be observed between the P4 and P9 sequences, and between the P5 and P10 sequences. N. M. Mansour et al. The BART superfamily FEBS Journal 274 (2007) 612–629 ª 2007 The Authors Journal compilation ª 2007 FEBS 621 [...]... binding of the ligand to the outside Such a scenario has been documented in the E coli phosphate-specific ABC transporter which interacts noncovalently with a sensor kinase (PhoR) to influence its activity [66] Fusion of the transporter domain to the sensor kinase domain suggests a close functional relationship between the two domains [58,67–69] The last family within the BART superfamily, the KPSH family,... be present in the cytoplasm, this suggests that the additional N-terminal TMSs probably have their N-termini in the cytoplasm If so, the conserved 5 TMS unit goes from out to in This would suggest that members of the RFT family, with 5 putative TMSs, may also have their N-termini outside and their C-termini inside Because the 5 TMS transporters show greatest sequence similarity with the first N-terminal... ligand through the membrane could actually be the sensed event that activates or inhibits the sensor kinase as in the case of phosphoryl transfer-dependent regulation via the E coli phosphoenolpyruvate-dependent phosphotransferase system [64,65] Third, the N-terminal domain might be both a sensor and a transporter, acting on the same ligand, but with the sensor function independent of the transport... superfamilies of transporters where transporter homologues serve as receptors, either while retaining their transport function, or while losing it [3,58– 63] In a few members of the sodium:solute symporter superfamily, full-length transporter domains are fused to sensor kinase domains [3] As for the SHK family, it is not known if the transporter domain is active as a transporter, or if it functions exclusively... families of the BART superfamily with no functionally characterized members One (UNK) includes members that look like typical 10 TMS porters The second (SHK) proved to be a coherent family of structurally similar proteins with an N-terminal 6 TMS transporter domain with TMSs 2–6 being homologous to the 5 TMS element that characterizes all members of the BART superfamily Because the C-termini of these proteins... hydropathy (top) and similarity (bottom) plots for the Acr3 family (B) The phylogenetic tree for the Acr3 family proteins The list of proteins and the multiple alignment upon which these plots were based can be found in Table S5 and Fig S5A on our website, respectively The bootstrapped trees are shown in Figs S5B and S5C The parsimony tree is shown in Fig S5D The phylogenetic tree shown in Fig 6B reveals... units in the 10 TMS homologues, we suggest that these proteins also display their N- and C-termini outside and their central loops inside These predictions were confirmed when we conducted charge distribution studies (data not presented) The positive inside rule [55–57] has provided valid predictions for transport protein topology Its application to members of the six families of the BART superfamily. .. for the UNK family (B) Phylogenetic tree for the UNK family proteins The list of proteins and the multiple alignment upon which these plots were based can be found in Table S6 and Fig S6A on our website, respectively The bootstrapped trees are shown in Figs S6B and S6C The parsimony tree is shown in Fig S6D FEBS Journal 274 (2007) 612–629 ª 2007 The Authors Journal compilation ª 2007 FEBS 623 The BART... single subcluster, as do the b-proteobacterial proteins, the c-proteobacterial proteins of cluster 10 fall into two subclusters, one for the Pseudomonads and one for the Xanthomonads These two c-proteobacterial genuses are known to be distantly related to each other Motif similarities among all 10 TMS homologues The C-terminal regions of the consensus sequences of TMSs 4 in the three 10 TMS families... sufficient to establish homology [1] The six families described above as well as the two repeat units of the 10 TMS proteins are therefore derived from a single ancestral sequence, and consequently, they comprise a single superfamily Discussion In addition to defining the phylogenetic and structural properties of the two previously recognized families (BASS and Acr3), and the newly discovered 5 TMS transport . and YKAA). The former targets the protein to the apical membrane in the absence of the latter, but the latter overrides the former, targeting the protein to the baso- lateral membrane [48]. The ileal. to each other than they are to any of the other sensor kinases, and only these have the homologous N-terminal hydrophobic domain com- mon to the RFT family proteins. The third domain is the HATPase-c. Fusion of the transporter domain to the sensor kinase domain suggests a close functional relationship between the two domains [58,67–69]. The last family within the BART superfamily, the KPSH