Department of Biology, University of California at San Diego, La Jolla, CA 92093-0116, USA1
Author for correspondence: Milton H. Saier, Jr. Tel: +1 858 534 4084. Fax: +1 858 534 7108. e-mail: msaier{at}ucsd.edu
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ABSTRACT |
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Keywords: amino acids, transport, evolution, superfamily, secondary carriers
Abbreviations: AAAP, amino acid/auxin permease; AAT, amino acid transporter; ABT, archaeal/bacterial transporter; ACT, amino acid/choline transporter; APA, basic amino acid/polyamine antiporter; APC, amino acid/polyamine/organocation; CAT, cationic amino acid transporter; EAT, ethanolamine transporter; GGA, glutamate:GABA antiporter; HAAAP, hydroxy/aromatic amino acid permease; LAT, L-type amino acid transporter; SGP, spore germination protein; TC, transporter classification; TMS, transmembrane -helical spanner; YAT, yeast amino acid transporter
a Present address: The Institute for Genomic Research (TIGR), 9712 Medical Center Drive, Rockville, MD 20850, USA.
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INTRODUCTION |
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One of the half-dozen largest of these superfamilies is the amino acid/polyamine/organocation (APC) superfamily [TC (transporter classification) no. 2.A.3; Closs et al., 1993 ; Reizer et al., 1993
; Saier, 1998
, 1999a
, b
, c
, 2000a
, b
; see also http://www-biology.ucsd.edu/~msaier/transport/]. This superfamily includes members that function as solute:cation symporters and solute:solute antiporters (Devés & Boyd, 1998
; Isnard et al., 1996; Kashiwagi et al., 1997
). Evidence suggests that in mammalian cells, simultaneous co- and counter-transport of ions with the amino acid can occur with a variety of complicated stoichiometries. For example, uptake of an amino acid can be accompanied by cotransport of three Na+ and one Cl- while being countertransported against a K+ (Beckman & Quick, 1998
).
The majority of homologous integral membrane APC transport proteins appear to exhibit a uniform topology with twelve transmembrane -helical spanners (TMSs) in a single polypeptide. This predicted topology has been experimentally verified for several bacterial homologues (Cosgriff & Pittard, 1997
; Ellis et al., 1995
; Hu & King, 1998a
). APC permease polypeptide chains vary in size from about 400 aminoacyl residues to about 800 residues (Sophianopoulou & Diallinas, 1995
). The larger proteins exhibit hydrophilic N- and C-terminal extensions as well as occasional enlarged inter-TMS loops. Proteins of one group, the LAT (L-type amino acid transporter) family (TC no. 2.A.3.8) derived from animals, are sometimes found in association with auxiliary proteins of the rBAT family (TC no. 8.A.9), and some of these same proteins, when defective, give rise to human diseases (Devés & Boyd, 1998
; Estévez et al., 1998
; Markovich et al., 1993
; Mastroberardino et al., 1998
; Palacín et al., 1998
; Sato et al., 1999
; Torrents et al., 1998
; Verrey et al., 1999
). Some of the animal homologues of the CAT (cationic amino acid transporter) family (TC no. 2.A.3.3) have been found to serve as viral receptors (Kim et al., 1991
; Reizer et al., 1993
; Wang et al., 1991
).
The substrate specificities of several APC superfamily permeases have been carefully studied revealing that while some have exceptionally broad specificity, others are restricted to just one or a few amino acids or related compounds (Brechtel & King, 1998 ; Hu & King, 1998b
, c
; Isnard et al., 1996
; Kashiwagi et al., 1997
; Sato et al., 1999
). One of these permeases, a histidine permease of Saccharomyces cerevisiae, surprisingly, has been implicated in manganese transport (Farcasanu et al., 1998
). Their regulatory properties have been the subject of investigations (see, for example, Sanders et al., 1998
).
No comprehensive phylogenetic analysis of the APC superfamily has been conducted since 1993, when only 14 members of the family were analysed (Reizer et al., 1993 ). Six years later, this superfamily has expanded to include 175 sequenced members that cluster in 10 defined families. Representation is found in all major domains of life (Paulsen et al., 1998a
, b
and unpublished results; see http://www-biology.ucsd.edu/~ipaulsen/transport/). A recent study has led to the suggestion that this superfamily is related to other currently recognized families of amino acid transporters, but this suggestion has yet to be rigorously established (Young et al., 1999
).
In this paper we update our earlier reports, providing structural, functional and phylogenetic descriptions of the proteins of the APC superfamily. All currently sequenced members of each of the 10 families that comprise this superfamily are tabulated, and their substrate specificities and modes of action, when known, are described. Phylogenetic tree construction reveals their evolutionary relationships to each other. Motif analyses provide sequence fingerprints for each of the 10 constituent families of the APC superfamily. The members of each of these families are also shown to exhibit uniform or nearly uniform topological features that prove characteristic of the superfamily as a whole. Distinctive properties that distinguish some of the different families are also noted. Thus, while all members of eight families appear to exhibit 12 TMSs, the members of one prokaryotic family [the SGP (spore germination protein) family] have 10 TMSs while the eukaryotic members of one of the ubiquitous families (the CAT family) have 14 TMSs. Further evidence is presented for a relationship of the APC superfamily to two other families of amino acid transporters.
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METHODS |
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RESULTS |
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The families vary in size from 4 members [the EAT (ethanolamine transporter) family; TC 2.A.3.5] to 36 members [the AAT (amino acid transporter) family; TC 2.A.3.1]. Other relatively large families include the YAT (yeast amino acid transporter) family with 29 members, the LAT family with 26 members, the CAT family with 21 members and the APA (basic amino acid/polyamine transporter) family with 20 members. All remaining families are relatively small, the largest being the ACT (amino acid/choline transporter) family with 13 members. For an introduction to the TC system of transport protein classification recently approved by the transport nomenclature panel of the International Union of Biochemistry and Molecular Biology, see Saier (1999c , 2000b
).
Of the 10 families, half of them are represented only in bacteria. One family, the ABT (archaeal/bacterial transporter) family, is found only in bacteria and archaea but not eukaryotes. The CAT family is found in bacteria and eukaryotes but not archaea. Finally, three families are found only in eukaryotes (the ACT, LAT and YAT families). While YAT family members are derived exclusively from the fungal kingdom (yeast, fungi and mushrooms), ACT family members are derived from both plants and fungi but not animals, and LAT family proteins occur in both fungi and animals but not plants. Thus, all animal proteins are included in only two families, all plant proteins are similarly included in just two families, but fungal proteins are included in three families. Four of the five largest families are those which have representation in eukaryotes, but the largest family is specific to bacteria. The four smallest families are prokaryote specific.
Without exception, the five families specific to bacteria include members with the smallest polypeptide chains. The smallest of these proteins are restricted to the SGP family (size range 329373 residues). No member of any other family is this small and this is the only family in which members have less than 12 putative TMSs. Proteins of the SGP family display just 10 TMSs. For the remaining four bacterial families, the protein size range is nearly the same (418556 residues). Surprisingly, the ABT family shows a size range of 422736 residues. Finally, the four families represented in eukaryotes show considerable size variation (440852 residues). One of these families, the CAT family, exhibits the unique characteristic that all of the eukaryotic members display 14 putative TMSs although the prokaryotic members of this same family display the more usual 12 TMSs. Thus, eight families include members that exhibit 12 TMSs, one family consists of proteins with either 14 or 12 TMSs and one includes members with 10 TMSs. Size and topological variation will be analysed in greater detail below.
Establishment of homology
Table 2 summarizes the results of the statistical analyses of interfamilial binary sequence comparisons. As discussed previously (Saier, 1994
, 1996
), a comparison score of 9 standard deviations (SD) for a stretch of at least 60 residues in comparable regions of two proteins is considered sufficient to establish homology. The odds that this degree of similarity could have arisen by chance is 1 in 10-19. We and others have published the arguments leading to the conclusion that a value of 9 SD could not have resulted either by chance or by a process of evolutionary sequence convergence (Doolittle, 1986
; Saier, 1994
). This conclusion is corroborated by the related functions and structures of the members of the APC superfamily. The results summarized in Table 2
therefore establish by these criteria that all 10 families listed in Table 1
include homologous proteins that are constituents of a single superfamily.
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The entries below the dashed line in Table 2 show that when SdaC of Escherichia coli was compared with TyrP of Chlamydia trachomatis, a comparison score of 11 SD was obtained. This value is sufficient to establish that these two proteins belong to a single family. Other comparisons reported in Table 2
, where members of the HAAAP family are compared with members of the APC and AAAP families, do not reach a value of 9 SD. The comparison scores therefore do not allow us to establish homology between these families. The values of 78 SD obtained, however, are sufficient to strongly suggest homology. We therefore suggest that the AAAP and HAAAP families are distant constituents of the APC superfamily, although we cannot prove this contention statistically.
Sequenced members of the APC superfamily
Table 3 shows the identified, sequenced members of the 10 established families that comprise the APC superfamily. The table presents the abbreviations of the proteins to be used in this study, the database descriptions of these proteins, their source organisms, their sizes, and their database accession numbers as well as their GenBank identifier (gi) numbers. Analyses of the sequences of the listed proteins provide the basis for the conclusions presented in this paper.
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The complete multiple alignments of the proteins that comprise each of the 10 families of the APC superfamily, upon which the average hydropathy plots shown in Fig. 2 were based, can be found at http://www-biology.ucsd.edu/~msaier/transport/phylo.html. These alignments were generated with the TREE program (Feng & Doolittle, 1990
). Examination of these multiple alignments reveals that in general, well-conserved regions in all of these alignments can be found in which some residues are fully conserved and many more are largely conserved. It is clear that the program used to generate these alignments has aligned the sequences correctly.
APC superfamily signature sequences
Table 4 presents signature sequences for the proteins of the 10 families within the APC superfamily. These sequences serve as identification motifs for these families. They were screened against the SWISS-PROT and TREMBL databases and retrieved only members of the represented family. By this criterion, they are bona fide signature sequences specific to these families. They can be used to identify additional members of these families as they are sequenced.
Several of the larger families in the APC superfamily are included within the Pfam (http://www.sanger.ac.uk/Software/Pfam/) database of protein families as the amino acid permease family (accession no. PF00324, prosite no. PDOC00191). However, the SGP family has been assigned to a distinct family (Pfam accession no. PB004126). Several of the smaller APC families are not included in the Pfam database (Bateman et al., 2000 ; Hofmann et al., 1999
).
Phylogeny of the 10 families within the APC superfamily
Figs 312 present phylogenetic trees for the proteins of the 10 families within the APC superfamily. Evaluation of these trees provides evidence regarding paralogous and orthologous relationships. Thus, if divergence has arisen solely due to speciation, the genes are orthologues, but if they arose as a result of gene duplication events within a single organism, they are paralogues. The expected relative distances for orthologues can be estimated based on relative phylogenetic distances observed for the 16S rRNAs from the organisms under consideration. It should be noted, however, that while most homologues in a single species can be assumed to be paralogues, it is more problematic to assign orthologous relationships for proteins from different species unless functional data are available. The reader should therefore be cognizant of this problem when considering conclusions based on the phylogenetic trees presented below.
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Fig. 4 shows the tree for the bacterial APA family. Four clusters (14) and two long branches (5 and 6) can be seen. The seven E. coli paralogues appear in all four primary clusters. Thus, three E. coli paralogues (PotE, YjdE and CadB) as well as a Haemophilus influenzae orthologue (PotE Hin), similar to PotE Eco, are found in cluster 1. In the large cluster 2, no two members are from the same organism. Thus, no paralogous pairs are found in this cluster. Nevertheless, these proteins are clearly not all orthologues. For example, the great distance between Yvs Bsu and ArcD Bli, derived from B. subtilis and Bacillus licheniformis, respectively, suggests that these two proteins diverged following an early gene-duplication event rather than as a result of speciation. Similarly, although seven of the nine proteins in cluster 2 have been annotated as ArcD proteins, the relative distances of these proteins do not generally correlate with the phylogenetic distances of the organisms. This fact suggests that they are not all orthologues of similar function. Cluster 3 consists of a probable pair of orthologues from E. coli and Aeromonas salmonicida, and cluster 4 consists of a possible orthologous pair from E. coli and B. subtilis (YkbA Bsu with either YhfM or Orf1 of E. coli). The two E. coli proteins in this cluster probably arose from a very recent gene-duplication event. Orf1 Sco (5) and PotE Rpr (6) are found at the ends of long branches. The great distance separating PotE Eco from PotE Rpr suggests that these two proteins are not orthologues. PotE Rpr may have been incorrectly annotated.
The tree shown in Fig. 5 for the CAT family shows clustering in accordance with the source organism. Thus, cluster 1 is specific for Gram-positive bacteria, clusters 2 and 3 include only mammalian members, cluster 4 is a group of three paralogues from the nematode Caenorhabditis elegans and cluster 5 includes two paralogues from the plant Arabidopsis thaliana. Fig. 6
(the ACT family) shows a group of yeast and fungal proteins related to one plant protein. Little clustering is observed except that Aap2 Ncr and Orf5 Spo are likely to be orthologues, and the clustering of YahB Spo, Orf3 Spo and Gpt1 Cal suggests similar functions for these proteins. The EAT family tree (Fig. 7
) is consistent with orthologous relationships between EntP Rer and EutP Zmo as well as between Orf1 Sme and Orf1 Mba.
The ABT family tree (Fig. 8) resembles the EAT family tree in that little clustering is observed. Neither the archaeal nor the bacterial proteins cluster together, suggesting that early gene duplication occurred before divergence of these two domains of prokaryotes. Alternatively, one could invoke lateral gene transfer to account for the results. The two B. subtilis paralogues probably arose by a relatively recent gene-duplication event.
In the GGA family tree (Fig. 9), the four E. coli paralogues are each found on distant branches. The XasA proteins of Chlamydia pneumoniae and Chl. trachomatis are undoubtedly orthologues, but the close clustering of XasA Eco and GadC Lla is surprising. Because E. coli and Lactococcus lactis are Gram-negative and Gram-positive bacteria, respectively, such close clustering would not be anticipated. We considered that this protein pair might provide an example of a recent lateral gene-transfer event between these two bacterial kingdoms. The same seemed possible for Orf54 Cpe and YgjI Eco which, however, are more distant from each other. Indeed, examination of the G+C contents of these genes revealed that the Gram-positive bacterial homologues exhibit the G+C values expected for the source organisms (35·0 mol% for the gadC gene of L. lactis compared to 35·4 mol% expected for L. lactis genes, and 30·8 mol% for the orf54 gene of Clostridium perfringens compared to 31·0 mol% expected for Clo. perfringens genes), but the E. coli genes deviated significantly from expectation based on the G+C content for E. coli genes as a whole (46·0 mol% for the xasA gene and 45·5 mol% for the ygjI gene compared to 51·4 mol% expected for E. coli genes). In fact, the G+C contents of the two E. coli genes are nearly the same at each of the three nucleotide positions of the codons in the E. coli xasA and ygjI genes, and they differed considerably from those expected for native E. coli genes, suggesting that they were obtained horizontally from the same organism or two very similar organisms. Codon usages for these two genes proved also to be more similar to each other than to E. coli genes (data not shown). These results suggest that E. coli acquired both of these genes from a Gram-positive bacterium long after Gram-positive and Gram-negative bacteria diverged from each other.
The phylogenetic tree for the large LAT family (Fig. 10), which includes animal and yeast proteins, reveals clustering patterns that in many cases reflect the phylogenies of the source organisms. Thus, the two yeast proteins, Mup1 and Mup3, cluster loosely together as do four ORFs from Cae. elegans. However, Cae. elegans proteins are found on two additional branches that cluster very loosely with the mammalian proteins. A number of mammalian orthologous and close paralogous relationships can be proposed on the basis of the tree configuration (see Fig. 10
).
The bacterial SGP tree (Fig. 11) reveals some early and late gene-duplication events as well as one probable orthologous relationship for the various established and putative germination proteins. Thus, GrkB Bsu and GerAB Bme are probable orthologues, but the cluster of three B. subtilis proteins at the bottom of the tree (GerAB, Orf1 and GerBB) represent paralogues that probably arose by recent gene-duplication events (Corfe et al., 1994
; Zuberi et al., 1987
). On the other hand, YfkT, GrkB and GerXB represent B. subtilis proteins that presumably arose by early gene-duplication events, maybe during the early origin of sporulation in the Bacillus line. This tree illustrates the tremendous amount of gene duplication that must have occurred in response to pressures arising during development of a program of prokaryotic differentiation. All of these proteins presumably arose by gene-duplication events in order to render spore germination responsive to the presence of amino acids in the growth medium.
Finally the YAT family (Fig. 12), where the vast majority of the proteins are from one species (Sac. cerevisiae), reveals a number of interesting orthologous and paralogous relationships. Particularly worthy of note is the huge cluster of 10 Sac. cerevisiae paralogues in a single cluster at the bottom of the tree. The occurrence of additional Sac. cerevisiae paralogues scattered throughout the tree is also noteworthy. Many of these yeast proteins are functionally characterized and they exhibit differing specificities. Thus, like bacilli, yeast has proliferated paralogues within the APC superfamily. Although many of these proteins are known to be active in the vegetative yeast cell, it will be interesting to determine if any of them function in the regulation of spore germination as has been demonstrated for the SGP family in Bacillus.
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DISCUSSION |
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Protein size and topological comparisons
Size and topological analyses showed that the SGP family (TC 2.A.3.9) includes the smallest proteins in the APC superfamily and these proteins display only 10 putative TMSs instead of the usual 12. The deficiency is at the C termini of these proteins. Interestingly, no member of this family has been shown to be a transporter, and the possibility that they serve as receptors rather than transporters can be entertained. Thus, loss of TMSs 11 and 12 may result in alteration of function. If, however, a transport function is demonstrated for one of these proteins, one can suggest that TMSs 11 and 12 in other APC superfamily permeases are not essential for transport.
One family, the CAT family, the only family to have representation in both prokaryotes and eukaryotes, shows a 12 TMS topology for the prokaryotic members but a 14 TMS topology for the eukaryotic members. Why these eukaryotic proteins, but no other members of the APC superfamily, have two extra putative TMSs is not known. Because the bacterial 12 TMS proteins cluster separately from the eukaryotic proteins on the CAT family tree (Fig. 5), it is reasonable to propose that the addition of two extra TMSs to the C termini of these proteins occurred only once during the evolution of the APC superfamily. It is possible that this C-terminal extension is related to the viral-receptor function of several of these proteins, although it could not have evolved to serve this function (see Reizer et al., 1993
).
Functionally uncharacterized families
No functional data are available for members of one of the APC superfamily families, the ABT family (TC 2.A.3.6). This family is represented in three archaea (Methanococcus jannaschii, Methanobacterium thermoautotrophicum and Archaeoglobus fulgidus) and three bacteria (E. coli, B. subtilis and Myc. tuberculosis). We can assume that these proteins transport amino acids and/or their derivatives, but direct experimentation will be necessary to establish this assumption. Similarly, although proteins of the SGP family (TC 2.A.3.9) have clearly been implicated in spore germination, there is no evidence as to whether these proteins function in transport or as transmembrane receptors, signalling the presence of amino acids to cytoplasmic regulatory proteins. Determination of the biochemical functions of these proteins will prove of interest for a more complete understanding of the mechanisms regulating endospore germination in Bacillus species.
Distant phylogenetic relationships
Although we were able to establish homology for all of the members of the APC superfamily included in this study, we also extended an earlier suggestion (Young et al., 1999) that two other recognized amino acid transporter families are distantly related to the APC superfamily (Table 2; Saier, 2000a
). Thus, the eukaryotic AAAP family and the prokaryotic HAAAP family are most likely distant constituents of the APC superfamily although maximal comparison scores of only 78 SD were obtained when interfamilial comparisons with any established member of the APC superfamily were done. The PSI-BLAST program with iterations (Altschul et al., 1997
), which uses a matrix/motif algorithm to detect distant phylogenetic relationships, also revealed similarities between members of these three families (unpublished results). It is therefore highly likely that the APC superfamily includes at least 12 recognized families instead of just 10, and because the AAAP family tree is complex, with several major sequence-divergent phylogenetic clusters (Young et al., 1999
), the AAAP family may appropriately be subdivided into more than one subfamily. However, because we prefer to adhere to a rigorous standard for establishment of homology, we will retain the AAAP and HAAAP families in the TC system separate from the APC superfamily. No evidence for a phylogenetic relationship between the APC superfamily and the many other families of transporters capable of transporting amino acids and their derivatives has been forthcoming (Saier, 2000a
).
It is interesting to note that members of the AAAP and HAAAP families exhibit 11 established or putative TMSs (see Young et al., 1999 for a summary of the biochemical and computational evidence). This number is different from that observed for any one of the established members of the APC superfamily. This fact suggests that the former two families may be more closely related to each other than to established members of the APC superfamily. Assuming all of these families to be related, we conclude that members of the extended APC superfamily may exhibit 10, 11, 12 or 14 TMSs. The functional implications of this finding have yet to be elucidated.
Concluding remarks
Although the reported studies have revealed that the APC superfamily is one of the largest superfamilies found in nature, it has apparently not diversified greatly in substrate specificity. All of its functionally characterized members transport amino acids and/or their derivatives. In view of the broad specificities of permeases in certain other superfamilies such as the larger major facilitator superfamily (TC 2.A.1) (Pao et al., 1998 ; Saier et al., 1999a
) or the smaller solute:sodium symporter family (TC 2.A.21), it is surprising that the APC superfamily has not diverged more extensively with respect to substrate recognition. An explanation for this observation is not currently at hand, but protein architectural constraints could be responsible (see Saier 1994
, 1996
, 1998
).
Future DNA sequencing projects and functional analyses of the revealed genes will undoubtedly result in both numerical and functional expansion of the 10 currently established families of the APC superfamily. Moreover, we can expect that completely novel families within the APC superfamily will be discovered. Evolutionary links to other families such as the AAAP family may also become unequivocally established as missing-link sequences become available. Additionally, more refined in silico tools for establishing homology as well as high resolution three-dimensional structural data for related secondary carriers may provide bases for establishing evolutionary links. We anticipate that the rules governing the evolutionary expansion of ancient superfamilies will soon become more clearly defined. Such advances will undoubtedly shed light on the most basic structural restrictions that determine the propensity for functional diversification of homologous transmembrane solute-transport systems during evolution.
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ACKNOWLEDGEMENTS |
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Received 19 October 1999;
revised 28 March 2000;
accepted 24 May 2000.