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Families of transmembrane transporters selective for amino acids and their derivatives

Department of Biology, University of California at San Diego, La Jolla, CA 92093-0116, USA¹

Tel: +1 858 534 4084. Fax: +1 858 534 7108. e-mail: msaier{at}ucsd.edu

Keywords: transmembrane transport, phylogeny, evolution, amino acids, polyamines

The information presented in this review was initially prepared for presentation at the FASEB meeting on amino acid transport held in Copper Mountain, Colorado, June 26–July 1, 1999 and was updated in January 2000 following the meeting of the Transport Nomenclature Panel of the International Union of Biochemistry and Molecular Biology (IUBMB) in Geneva, November 28–30, 1999. The system of classification described in this review reflects the recommendations of that panel.

	Overview

TOP Overview Background Characteristics of transporters... Most amino acid transporters... ABC (ATP-binding cassette) -type... Secondary carriers Secondary amino acid transporter... Amino acid transporter families... Families of secondary... Families of channel-forming... Families of peptide transporters Protein-transport systems Conclusion REFERENCES

 
Twenty-one families of secondary [proton-motive force (PMF)-driven] carriers are herein described, which, in addition to 13 of the currently recognized 48 families within the ATP-dependent ATP-binding casette (ABC) superfamily and three families of channel proteins, largely account for the transport of amino acids and their derivatives (excluding proteins) into and out of all living cells on Earth. Family characteristics as well as structural and functional properties of the family constituents are described. By reference to our web site (http: / / www-biology.ucsd.edu /~msaier / transport /), sequences, phylogenetic relationships, detailed substrate-specificity information and the distribution of members of each family in 20 fully sequenced genomes of bacteria, archaea and eukaryotes are available. This review provides a comprehensive guide to the diversity of carriers that mediate the transport of amino acids and their derivatives across cell membranes.

	Background

TOP Overview Background Characteristics of transporters... Most amino acid transporters... ABC (ATP-binding cassette) -type... Secondary carriers Secondary amino acid transporter... Amino acid transporter families... Families of secondary... Families of channel-forming... Families of peptide transporters Protein-transport systems Conclusion REFERENCES

 
Amino acids are the building blocks of proteins. They also provide raw materials for a variety of important cellular processes such as energy generation, nitrogen metabolism, cell-wall synthesis, antibiotic production and intercellular communication. Amino acids can be zwitterionic, anionic or cationic, and their side chains can be hydrophobic, semipolar or strongly hydrophilic. These side chains may be aliphatic or aromatic, and they sometimes serve as physiologically important proton donors and acceptors in enzymes with pK_a values ranging from about 4 to 12. Amino acids in proteins can be derivatized by methylation, acetylation, formylation and phosphorylation; the free compounds can be decarboxylated to amines, and L-aminoacyl derivatives can be condensed enzymically with other L-amino acids, with amino acids of the D configuration and with hydroxy acids to yield peptides and depsipeptides of unusual composition. These aminoacyl residue derivatives serve a wide variety of biological functions including those of biological warfare, quorum sensing, intercellular signalling and regulation of gene expression. It is therefore not surprising that several transporter families have evolved to control and facilitate exchange of these substances between cells and their environment, between cells and their organelles, and between cells and other living entities.

Over two dozen transporter families and superfamilies are currently recognized that include members that transport amino acids and their derivatives (Paulsen et al., 1998a , b ; Saier, 1998 , 1999a , b , c ; see also http://www-biology.ucsd.edu/~msaier/transport/). A number of interesting questions arise related to these families. For example (1) what are the ranges of substrates transported by members of each of these families? (2) What energy-coupling mechanisms do they employ to accumulate or expel their substrates within or from living cells? (3) What is/are the polarity/polarities of transport allowed by members of each such family? (4) In what range of organisms are members of each of these families found? (5) When, during evolutionary history, did each family appear and via what route? (6) What are the mechanisms used to effect transmembrane transport? (7) Do homologues of these transport proteins function in capacities other than in transport, and if so, in what capacities? (8) Are certain families concerned primarily with a particular physiological function (e.g. uptake, secretion or intercellular communication), or do they all serve comparable and broadly overlapping physiological functions? (9) How are the syntheses, activities, intracellular localizations and proteolytic degradation processes of transporter proteins regulated, and in response to what stimuli? (10) What transport characteristics are readily altered during evolution and which characteristics remain relatively immutable? These and other questions will be systematically addressed in the following section, and information that will allow the reader to personally evaluate the answers to these questions, relevant to specific families, will be provided with greater insight in the subsequent sections.

	Characteristics of transporters and their homologues

TOP Overview Background Characteristics of transporters... Most amino acid transporters... ABC (ATP-binding cassette) -type... Secondary carriers Secondary amino acid transporter... Amino acid transporter families... Families of secondary... Families of channel-forming... Families of peptide transporters Protein-transport systems Conclusion REFERENCES

 
Careful examination of the properties of the functionally characterized members of currently recognized transporter families selective for amino acids and their derivatives reveals tentative answers to the ten questions posed above.

(1) The range of substrates transported by an individual member of a family can be narrow or broad, depending on the transporter, and an entire family can often transport a wide variety of structurally related compounds. Seldom, however, do members of a single family catalyse the transport of structurally divergent types of compounds (i.e. sugars versus amino acids versus inorganic anions).

(2) Only two forms of energy are generally used for the active uptake or expulsion of amino acids and their derivatives: electrochemical energy stored in the gradients of H⁺, Na⁺ and structurally related substrates (utilized by secondary active carriers), and chemical energy in the form of ATP (utilized by ABC-type uptake and efflux systems as well as by several macromolecular transport systems). Channel proteins usually do not couple transmembrane translocation to energy, although a few interesting exceptions are known.

(3) With very few exceptions (i.e. the major-facilitator superfamily and the ABC superfamily) all members of a phylogenetically defined family function with strongly preferential inwardly directed or outwardly directed polarity. Seldom do different members of a single transporter family transport solutes with opposite polarity, and no permease is currently known to actively take up its substrates under one set of experimental conditions but actively extrude them under another set of conditions.

(4) Some families of sequence-related transport proteins are found ubiquitously in all major groups of organisms (i.e. among the bacteria, archaea and eukaryotes), while others are restricted to just one of these three domains. In some cases, all members of a family derive from just one kingdom within one of these domains. The former proteins may represent ancient families of transporters while the latter proteins are those which have more recently appeared, either de novo or by extensive sequence divergence from a pre-existing family.

(5) As noted above and discussed in several reviews (Saier, 1994 , 1996 , 1998 ), it appears that new families have appeared throughout evolutionary history although with variable frequencies. Moreover, they probably underwent sequence divergence at rates that were neither constant with time nor constant for all members of the family. Most of the families that appeared after bacteria, archaea and eukaryotes separated from each other during the ‘great split’ are apparently still restricted to the domain in which they arose. These proteins have often arisen from much simpler peptides or proteins as a result of internal gene-duplication and gene-fusion events. Little lateral transfer of the resultant genetic information between the three domains of organisms has occurred, at least during the past two billion years.

(6) The primary modes of transport have historically been thought to involve, and are still considered to involve, channels and carriers. While the mechanistic details of the former type of transporters are well understood, in part due to the availability of high-resolution three-dimensional structural data for these proteins, carrier mechanisms are still poorly understood (West, 1997 ). These carrier mechanisms represent the primary types responsible for the transmembrane translocation of organic nutrients.

(7) Homologues of transporters almost never function in a primary capacity other than transport. Those that do usually serve as simple receptors, influencing a single aspect of cellular physiology. We have consequently proposed that integral transmembrane transporters evolved as a distinct class of proteins, and that they did not appear by modification of other protein types (i.e. enzymes, structural proteins or regulatory proteins) (Saier & Tseng, 1999 ).

(8) Some families or subfamilies of transporters selective for amino acids and their derivatives seem to have evolved to serve a highly specific physiological function such as that of auxin secretion for the purpose of promoting gravitropism in plants, performed by members of the auxin efflux carrier (AEC) family [transporter classification (TC) no. 2.A.69], or that of promoting dormant spore germination in Bacillus species, performed by members of the spore germination protein (SGP) family (TC no. 2.A.3.9) of the amino acid/polyamine/organocation (APC) superfamily (TC no. 2.A.3). Other families selectively catalyse either uptake or efflux of specific amino acids and/or their derivatives for the purpose of nutrition, cellular protection or intercellular signalling. Thus, it is clear that members of a family may have evolved to provide specific physiological functions. Nevertheless, specialization for a specific physiological function appears to be a late-evolving characteristic and therefore is of minimal importance for purposes of classifying transporters (Saier & Tseng, 1999 ).

(9) In general, the syntheses, activities and degradation of transport proteins are stringently regulated, employing the same or similar universal mechanisms (i.e. ligand binding, covalent modification, protein–protein interactions) that are operative and well established for cytoplasmic enzymes. Similarity, subcellular localization is determined by targeting sequences in the proteins of both prokaryotes and eukaryotes, and by complex, reversible, hormone-regulated processes in higher eukaryotes. These topics are in general beyond the scope of this review but are the topics of numerous other reviews, to which the reader is referred (Saier et al., 1989 ; Knutson, 1991 ; Fekkes & Driessen, 1999 ; Koch et al., 1999 ).

(10) Finally, the classification and characterization of transporter families have revealed that transport mode and energy-coupling mechanism are highly conserved evolutionary traits, that protein topology, polarity of transport and substrate specificity are traits conserved to an intermediate degree, and that the regulatory mechanisms imposed on transporters are late evolving, poorly conserved traits. The utilization of these various traits for purposes of transport-protein classification has been the topic of discussion in several recently published reviews (Saier, 1999a , b , c ; Saier & Tseng, 1999 ).

Below, the transporter families concerned with transmembrane transport of amino acids and their derivatives are discussed. Groups of functional types will first be considered according to class and transport mechanism, and the unique features of each such family will be presented.

	Most amino acid transporters are secondary carriers

TOP Overview Background Characteristics of transporters... Most amino acid transporters... ABC (ATP-binding cassette) -type... Secondary carriers Secondary amino acid transporter... Amino acid transporter families... Families of secondary... Families of channel-forming... Families of peptide transporters Protein-transport systems Conclusion REFERENCES

 
We currently recognize about 120 families of channel/pore proteins, about 30 families of primary active transport systems and about 80 families of secondary carriers (Saier, 1998 , 1999a , b , c ; see also http://www-biology.ucsd.edu/~msaier/transport/). Additionally, dozens of families include transporters with undefined, or ill-defined, transport mechanisms. Of the former families, members of only one recognized family of channel proteins, the phospholemman (PLM) family (TC no. 1.A.27; Kirk & Strange, 1998 ), in addition to nonspecific outer-membrane porins of Gram-negative bacteria and eukaryotic organelles (TC classification division 1.B), and only one recognized family of primary carriers, the ABC superfamily (Saurin et al., 1999 ), can transport amino acids and their structurally related derivatives. All other families apparently consist of secondary carriers that function by PMF or SMF (sodium-ion motive force) -driven uptake, by PMF-driven efflux, by solute–solute exchange or by uniport. Secondary carriers will therefore be the main focus of this review.

	ABC (ATP-binding cassette) -type transporters

TOP Overview Background Characteristics of transporters... Most amino acid transporters... ABC (ATP-binding cassette) -type... Secondary carriers Secondary amino acid transporter... Amino acid transporter families... Families of secondary... Families of channel-forming... Families of peptide transporters Protein-transport systems Conclusion REFERENCES

 
ABC-type transporters fall into 48 currently recognized families within the ABC superfamily (Table 1). Of these, 19 include members that are exclusively uptake systems (all found in prokaryotes); 19 include members that are prokaryotic-specific efflux systems and 10 include members that are primarily eukaryotic-specific efflux systems. The uptake and efflux systems cluster separately on the phylogenetic tree for this superfamily (Saurin et al., 1999 ).

Of the prokaryotic ABC-type exporter families, three are concerned with drug efflux, five catalyse peptide export and two function in protein transport. The five peptide-exporter families are the Pep1–3E families (TC 3.A.1.101–103) and the two microcin-exporter families (the McbE and McjD families; TC 3.A.1.106 and 108 respectively), but some of the drug-export permeases may be capable of transporting certain peptides. Of the currently recognized eukaryotic families, two function primarily in peptide export (a-factor sex pheromone exporter, STE, and MHC peptide transporter, TAP; TC 3.A.1.206 and 209 respectively) and three function to export drugs (including some peptides). However, no eukaryotic ABC exporter is known to transport proteins. Eukaryotes export macromolecules by exocytosis rather than by a permease-mediated molecular-transport process. Thus, the functional diversity of prokaryotic ABC transporters far exceeds that of the eukaryotic ABC transporters. Of the 48 recognized families, 13 (27%) are concerned primarily with the transport of amino acids and their derivatives including peptides, and only two of these families derive from eukaryotes. In bacteria, another two families are concerned with protein export.

	Secondary carriers

TOP Overview Background Characteristics of transporters... Most amino acid transporters... ABC (ATP-binding cassette) -type... Secondary carriers Secondary amino acid transporter... Amino acid transporter families... Families of secondary... Families of channel-forming... Families of peptide transporters Protein-transport systems Conclusion REFERENCES

 
Table 2 tabulates the secondary carrier families that are known to function in transport of (a) amino acids and their conjugates, (b) amines, amides and polyamines and (c) peptides. Altogether, 19 families fall into category a, eight fall into category b, and four fall into category c. Nevertheless, there is a total of only 21 families. All families that fall into category b are also represented in category a and only two families of peptide transporters (oligopeptide transporter, OPT, and peptide-uptake permease, PUP) have not been shown to include members that transport simple amino acids. Below we examine these families in greater detail.

	Secondary amino acid transporter families found exclusively in bacteria

TOP Overview Background Characteristics of transporters... Most amino acid transporters... ABC (ATP-binding cassette) -type... Secondary carriers Secondary amino acid transporter... Amino acid transporter families... Families of secondary... Families of channel-forming... Families of peptide transporters Protein-transport systems Conclusion REFERENCES

The 10 families listed in Table 3 will be discussed individually below. The descriptions provided reflect our knowledge of these families as of July 1999.

The Dcu (C₄-dicarboxylate uptake) family (TC 2.A.13)
Several proteins of the Dcu family have been sequenced, all from Gram-negative bacteria (Engel et al., 1994 ; Six et al., 1994 ; Unden & Bongaerts, 1997 ; Golby et al., 1998 ). The two best characterized systems (DcuA and DcuB) are from Escherichia coli. The fully sequenced proteins of the Dcu family are of fairly uniform size (434–446 residues). They possess 12 putative TMSs, but DcuA of Escherichia coli has 10 experimentally determined TMSs with both the N and C termini localized to the periplasm. For DcuA, the ‘positive inside’ rule (von Heijne, 1992 ) is obeyed and two putative TMSs are localized to a cytoplasmic loop between TMS 5 and 6 and in the C-terminal periplasmic region.

The Escherichia coli DcuA and DcuB proteins transport aspartate, malate, fumarate and succinate, and function as antiporters with any two of these substrates. The two proteins exhibit 36% identity with 63% similarity, and both transport fumarate in exchange for succinate with about the same affinity (30 µM). Since DcuA is encoded near the gene for aspartase, and DcuB is encoded in an operon with the gene for fumarase, their physiological functions have been hypothesized to involve aspartate:fumarate and fumarate:malate exchange during the anaerobic utilization of aspartate and fumarate, respectively. However, the electroneutral antiport of fumarate for succinate during anaerobic fumarate respiration has been demonstrated, and both permeases are induced under anaerobic conditions and subject to catabolite repression. The two transporters can apparently substitute for each other under certain physiological conditions. A third dicarboxylate transporter, DcuC, exhibits slight sequence similarity with DcuA (optimized comparison score of 5 SD; 23% identity for a segment of 100 residues). This degree of similarity is insufficient to establish homology (Saier, 1994 , 1996 ) and consequently, DcuC is assigned to a distinct family (TC 2.A.61).

The BCCT (betaine/carnitine/choline transporter) family (TC 2.A.15)
Proteins of the BCCT family are found in Gram-negative and Gram-positive bacteria (Lamark et al., 1991 ; Eichler et al., 1994 ; Kappes et al., 1996 ; Kempf & Bremer, 1998 ; Peter et al., 1998 ). Their common functional feature is that they all transport molecules with a quaternary ammonium group [R-N⁺(CH₃)₃]. BCCT family proteins vary in length between 481 and 677 aminoacyl residues, possess 12 putative TMSs and are energized by PMF-driven proton symport.

The alanine/glycine:cation symporter (AGCS) family (TC 2.A.25)
Members of the AGCS family transport alanine and/or glycine in symport with a monovalent cation, Na⁺ and/or H⁺ (see Reizer et al., 1994 ). The proteins are 445–542 aminoacyl residues in length and possess 8–12 putative TMSs. They are found in Gram-positive and Gram-negative bacteria. Only two members of the family have been functionally characterized. These proteins show minimal sequence similarity to members of the APC family (TC 2.A.3; see below; Jack et al. 2000 ), but the significance of this observation is not known.

The branched chain amino acid:cation symporter (LIVCS) family (TC 2.A.26)
Characterized members of this family transport all three of the branched chain aliphatic amino acids [leucine (L), isoleucine (I) and valine (V)] (Reizer et al., 1994 ; Stucky et al., 1995 ; Tauch et al., 1998 ). They are found in Gram-negative and Gram-positive bacteria and function by a Na⁺ or H⁺ symport mechanism. They possess about 440 aminoacyl residues and display 12 putative TMSs.

The glutamate:Na⁺ symporter (GltS) family (TC no. 2.A.27)
A single member of this family has been functionally characterized (Deguchi et al., 1990 ). This permease (GltS of Escherichia coli) catalyses glutamate:Na⁺ symport. It exhibits 401 aminoacyl residues with 12 putative helical TMSs. Homologues are found in other bacteria including Haemophilus influenzae, Helicobacter pylori and Synechocystis.

The HAAAP (hydroxy and aromatic amino acid porter) family (TC 2.A.42)
The HAAAP family includes three well characterized aromatic amino acid:H⁺ symport permeases of Escherichia coli: a high-affinity tryptophan-specific permease, Mtr, a low affinity tryptophan permease, TnaB, and a tyrosine-specific permease, TyrP (Wookey & Pittard, 1988 ; Sarsero et al., 1991 ; Sarsero & Pittard, 1995 ). These proteins possess 403–405 aminoacyl residues with 11 TMSs. The HAAAP family also includes two well characterized permeases specific for hydroxy amino acids: one is the serine permease, SdaC, of Escherichia coli, the other is the threonine permease, TdcC, of Escherichia coli (Goss et al., 1988 ; Shao et al., 1994 ). These permeases are 429 and 443 aminoacyl residues long, respectively, and each possesses 11 putative TMSs. Homologues are present in a variety of Gram-negative bacteria. They show topological features common to members of the eukaryotic amino acid/auxin permease (AAAP) family (TC 2.A.18), and they display limited sequence similarity with some of them (Young et al., 1999 ). Since members of the AAAP family show limited sequence similarity with members of the large APC superfamily (TC no. 2.A.3) (Young et al., 1999 ; Jack et al., 2000 ), all of these proteins are probably related.

The TRAP-T (tripartite ATP-independent periplasmic transporter) family (TC 2.A.56)
TRAP-T family permeases generally consist of three components (Forward et al., 1997 ; Rabus et al., 1999 ). The best characterized of these systems is the DctMQP system of Rhodobacter capsulatus (Forward et al., 1997 ). DctM is a typical 12 TMS protein with weak sequence and motif similarity to several other families of secondary carriers (Rabus et al., 1999 ). However, DctQ is a 4 TMS integral membrane protein, and DctP is a periplasmic binding protein. Database searches and phylogenetic analyses have revealed that unlike most of the other families included in Table 3, homologous TRAP-T systems are found in archaea as well as bacteria. In some of these permeases, including all currently recognized archaeal systems, the M homologue is fused to the Q homologue yielding a 16 TMS protein, and in a few cases, the Q homologue is fused to the P homologue. Thus, in the TRAP-T family, as for the ABC superfamily, domain fusion/splicing has occurred repeatedly during evolution of the family.

All three Dct proteins are required for the uptake of dicarboxylates in Rhodobacter capsulatus, suggesting that these subunits function together, employing a concerted mechanism. Moreover, phylogenetic analyses reveal that all TRAP-T systems probably depend on the structural equivalent of the three proteins, DctMQP. The family appears to be an ancient but functionally unified family that evolved in parallel with the ABC superfamily, with little or no shuffling of constituents between families (Rabus et al., 1999 ). The TRAP-T family is the only family currently known for which an extracytoplasmic receptor functions in conjunction with a secondary carrier.

Many members of the TRAP-T family are functionally uncharacterized. An operon encoding a Synechocystis system includes a protein homologous to the glutamine-binding protein of an Escherichia coli ABC-type permease, and biochemical evidence has suggested that a glutamate transporter from Rhodobacter sphaeroides is a periplasmic binding protein-dependent, PMF-dependent, secondary carrier (Jacobs et al., 1996 ). Escherichia coli homologues may be involved in the uptake of pentoses and/or pentitols (Reizer et al., 1996 ; Sanchez et al., 1994 ). Thus, members of the TRAP-T family of permeases may take up widely divergent compounds.

The LysE (L-lysine exporter) family (TC 2.A.75)
One member of the LysE family (LysE of Corynebacterium glutamicum) is functionally well characterized, but functionally uncharacterized or partially characterized homologues are encoded within the genomes of a variety of bacteria and archaea (Bröer & Krämer 1991a , b ; Vrljic et al., 1996 , 1999 ; Zakataeva et al., 1999 ). All of these proteins are 190–240 aminoacyl residues in length and possess six hydrophobic regions. PhoA fusion analyses provided evidence that LysE of Corynebacterium glutamicum exhibits a 5 TMS topology, with the N terminus inside and the C terminus outside (Vrljic et al., 1999 ). LysE appears to catalyse unidirectional efflux of L-lysine and other basic amino acids such as L-arginine and L-ornithine, and it provides the sole route for L-lysine excretion in this bacterium. The energy source is believed to be the PMF (H⁺ antiport or OH^- symport).

The RhtB (resistance to homoserine and threonine) family (TC 2.A.76)
Distant homologues of LysE have been shown to expel threonine. They may catalyse efflux of homoserine and homoserine lactones as well (Aleshin et al., 1999 ; Zakataeva et al., 1999 ). Because homoserine lactones are signalling molecules in Gram-negative bacteria, this observation has special significance (Fuqua et al., 1996 ; Swift et al., 1996 ). The family that includes these amino acid efflux pumps has been termed the RhtB family, and in the transporter-classification system, it was given the TC number 2.A.76. Evidence has been presented that it and the LysE family (above), together with the cadmium-resistance (CadD) family (TC no. 2.A.77), compose a single superfamily, the LysE superfamily (Vrljic et al., 1999 ). All characterized members of this superfamily catalyse solute export, presumably by a proton-antiport mechanism.

The CAAT (carboxylate/amino acid/amine transporter) family (TC 2.A.78)
The CAAT family is a large family of integral membrane proteins with sizes ranging from 287 to 310 aminoacyl residues and exhibiting 10 putative TMSs. These proteins are derived from phylogenetically divergent bacteria and archaea, and Escherichia coli, Bacillus subtilis and Aspergillus fulgidus have multiple paralogues. With one PSI-BLAST iteration, they show low degrees of sequence similarity with members of the ubiquitous L-rhamnose transporter (RhaT) family (TC 2.A.9) and with the eukaryotic triose phosphate transporter (TPT) family (TC 2.A.50), found in plant chloroplasts and plastids as well as in yeast. One distant plant homologue is the Medicago nodulin N21-like protein (gbAC004218; 374 aa) of Arabidopsis thaliana.

Proteins of the CAAT family evidently arose by an internal gene-duplication event as the first halves of these proteins are homologous to the second halves. Although none of these prokaryotic proteins is functionally characterized, several members of the CAAT family have been implicated in solute transport. Thus, the MttP protein of the archaeon Methanosarcina barkeri may transport methylamine (Ferguson & Krzycki, 1997 ) and the YtfF protein of Chlamydia trachomatis may transport basic amino acids (Stephens et al., 1998 ). Additionally, MadN is encoded within the malonate utilization operon of Malonomonas rubra and PecM is encoded within a locus of Erwinia chrysanthemi controlling pectinase, cellulase and blue-pigment production. MadN may be an acetate-efflux pump while PecM might export the pigments produced by gene products encoded in the pecM operon (Berg et al., 1997 ; Reverchon et al., 1994 ).

	Amino acid transporter families that have been found in eukaryotes

TOP Overview Background Characteristics of transporters... Most amino acid transporters... ABC (ATP-binding cassette) -type... Secondary carriers Secondary amino acid transporter... Amino acid transporter families... Families of secondary... Families of channel-forming... Families of peptide transporters Protein-transport systems Conclusion REFERENCES

 
While Table 3 lists the properties of the ten families of amino acid transporters found exclusively in prokaryotes, Table 4 provides comparable information for the nine families that occur in eukaryotes. Of these families, the AAAP and mitochondrial carrier (MC) families are found only in eukaryotes. The MC family undoubtedly arose in eukaryotes in response to a need for a new type of communication between the matrix of the mitochondrion and the cytoplasm of the eukaryotic cell (Kuan & Saier, 1993 ; Saier, 1994 , 1996 ). However, we have presented evidence (Young et al., 1999 ) that the AAAP family is distantly related to the ubiquitous APC superfamily, and this claim has recently been further substantiated (Jack et al., 2000 ).

Below, the nine families described in Table 4 are discussed.

The APC (amino acid/polyamine/organocation) family (TC 2.A.3)
The APC family of transport proteins includes nearly 250 currently sequenced members that function as solute:cation symporters and solute:solute antiporters (Closs et al., 1993 ; Reizer et al., 1993a ; Sophianopoulou & Diallinas, 1995 ; Isnard et al., 1996 ; Kashiwagi et al., 1997 ; Brechtel & King, 1998 ; Hu & King, 1998a , b ; Sanders et al., 1998 ; Sato et al., 1999 ). All functionally characterized members thus catalyse active solute uptake or exchange transport. These proteins occur in bacteria, archaea, yeast, fungi, unicellular eukaryotic protists, plants and animals, and are thus essentially ubiquitous. They vary dramatically in length, being as small as 350 residues and as large as 800 residues. The smaller proteins are generally of prokaryotic origin while the larger ones are of eukaryotic origin. Most of them possess 12 TMSs, but predicted topologies of 10 or 14 TMSs are also sometimes observed (Cosgriff & Pittard, 1997 ; Hu & King, 1998c ; Jack et al., 2000 ). The larger eukaryotic proteins have N- and C-terminal extensions that may serve regulatory functions. At least some animal proteins such as ASUR4 (gbY12716) and SPRM1 (gbL25068) associate with a type 1 transmembrane glycoprotein that is essential for insertion, stability or activity of the permease and forms a disulfide bridge with it (Verrey et al., 1999 ). These glycoproteins include the CD98 heavy-chain protein of Mus musculus (gbU25708) and the 4F2 cell surface antigen heavy chain of man (Mastroberardino et al., 1998 ). These homologues are members of the rBAT family of mammalian transporter-accessory proteins (TC 8.A.9). All of the members of the rBAT family are believed to associate with members of the L-type amino acid transporter (LAT) family (TC 2.A.3.8) within the APC superfamily to form heterodimeric complexes.

One APC family member, Hip1 of Saccharomyces cerevisiae, which clearly functions in amino acid transport, has also been implicated in heavy metal transport (Farcasanu et al., 1998 ). Another member of the family, Ssy1 of Saccharomyces cerevisiae, appears to be a transcriptional regulatory sensor (Didion et al., 1998 ). It is possible, but not demonstrated that APC family homologues in Bacillus subtilis that promote spore germination may function as receptors rather than transporters. Thus, a few APC family members may serve functions other than that of the transport of amino acids and their derivatives.

The AAAP (amino acid/auxin permease) family (TC 2.A.18)
The AAAP family includes over four dozen sequenced proteins from plants, animals, yeast and fungi (Fischer et al., 1995 ; Bennett et al., 1996 ; Rentsch et al., 1996 ; McIntire et al., 1997 ; Young et al., 1999 ). Individual permeases of the AAAP family transport auxin (indole-3-acetic acid), -aminobutyric acid, a single L-amino acid or multiple amino acids. Some of these permeases exhibit very broad specificities, transporting all of the L-amino acids naturally found in proteins. There are 16 AAAP paralogues in Saccharomyces cerevisiae, 13 in Caenorabditis elegans and at least 9 in Arabidopsis thaliana. These proteins, all from eukaryotes, vary from 376 to 713 aminoacyl residues in length, but most are 400–500 residues. Most of the size variation occurs as a result of the presence of long N-terminal hydrophilic extensions in some of the proteins, but size variation in the loops and the C termini is sometimes observed. These proteins exhibit 11 putative TMSs and show limited sequence similarity with members of the large APC superfamily. Thus, the AAAP family is probably part of the APC superfamily (Jack et al., 2000 ).

The major facilitator superfamily (MFS) (TC 2.A.1)
The MFS is a very old, large and diverse superfamily that includes over a thousand sequenced members (Pao et al., 1998 ; Saier et al., 1999b ). These permeases catalyse uniport, solute:cation (H⁺ or Na⁺) symport and/or solute:H⁺ or solute:solute antiport. Most are of 400–600 aminoacyl residues in length and possess either 12 or 14 putative or established TMSs. They exhibit specificity for sugars, polyols, drugs, neurotransmitters, Krebs-cycle metabolites, phosphorylated glycolytic intermediates, amino acids, peptides, osmolites, nucleosides, organic anions, inorganic anions, iron siderophores, etc. They are found ubiquitously in all three domains of living organisms. Each of the 29 currently recognized families is in general specific for one class of compounds (see http://www-biology.ucsd.edu/~msaier/transport/). While one family in the MFS includes permeases that catalyse amino acid (proline) uptake [the metabolite:H⁺ symporter (MHS) family (TC 2.A.1.6)], another, the peptide-acetyl-CoA transporter (PAT) family (TC 2.A.1.25), functions in peptide uptake in bacteria (see below) but in acetyl-CoA:CoA antiport in the endoplasmic reticular membrane of higher eukaryotes.

The proton-dependent oligopeptide transporter (POT) family (TC 2.A.17)
The POT family [also called the PTR (peptide transport) family] is primarily a peptide-transporter family, but one member is a nitrate/chlorate permease, and one has been reported to transport histidine as well as nitrate and peptides (Frommer et al., 1994 ; Zhou et al., 1998 ). Because this family is primarily concerned with peptide transport, it will be described below in the section entitled ‘families of peptide transporters’.

The solute:sodium symporter (SSS) family (TC 2.A.21)
All functionally characterized members of the SSS family catalyse solute uptake via Na⁺ symport (Reizer et al., 1994 ). The solutes transported may be sugars, amino acids, nucleosides, inositols, vitamins, urea or anions, depending on the transport system (Eskandari et al., 1997 ; Prasad et al., 1998 ; Sarker et al., 1997 ). The broad substrate specificity but uniform energy-coupling mechanism exhibited by members of this family are particularly unusual and noteworthy traits of a single family.

Members of the SSS family have been identified in bacteria, archaea and animals. They vary in size from about 400 residues to about 700 residues and possess 12–15 putative TMSs, often sharing a core of 13 TMSs. A 13 TMS topology with a periplasmic N terminus and a cytoplasmic C terminus has been experimentally determined for the proline:Na⁺ symporter, PutP, of Escherichia coli (Turk & Wright, 1997 ; Jung et al., 1998 ).

The neurotransmitter:sodium symporter (NSS) family (TC 2.A.22)
Members of the large NSS family catalyse uptake of a variety of neurotransmitters, amino acids, osmolytes and related nitrogenous substances by a solute:Na⁺ symport mechanism (Beckman & Quick, 1998 ; Kavanaugh, 1998 ; Berfield et al., 1999 ). Sometimes Cl^- is co-transported, and some of the NSS family members exhibit a K⁺ dependency. For example, human dopamine, glutamate and -aminobutyric acid transporters co-transport their positively charged or zwitterionic substrates with 2 or 3 Na⁺, and 1 Cl^- is probably co-transported, at least with dopamine and -aminobutyric acid. The glutamate system may counter-transport K⁺ (Clark & Amara, 1993 ). Most sequenced members of the NSS family are from animals, and these proteins are generally 600–700 aminoacyl residues in length with 12 TMSs. Bacterial and archaeal homologues have been sequenced, but few functional data are available for these proteins.

Recently, several members of the NSS family have been shown to exhibit channel-like properties under certain experimental conditions. Sizeable unitary ionic currents have been reported for membrane patches containing either the -aminobutyrate, noradrenaline or serotonin transporter (Galli et al., 1998 ). Channel-like currents have also been measured for mammalian Na⁺/H⁺/K⁺-coupled glutamate transporters of the dicarboxylate/amino acid:cation symporter (DAACS) family (TC no. 2.A.23; see below). Evidence suggests that these channels can accommodate neurotransmitters as well as inorganic ions. These observations suggest that, as has been demonstrated for carriers of a few other families, neurotransmitter transporters can be manipulated to function as voltage-gated channels. Whether or not this observation is of physiological relevance has yet to be determined.

The DAACS [dicarboxylate/amino acid:cation (Na⁺ or H⁺) symporter] family (TC 2.A.23)
Members of the DAACS family catalyse Na⁺ and/or H⁺ symport together with (a) a Krebs-cycle dicarboxylate (malate, succinate, or fumarate), (b) a dicarboxylic amino acid (glutamate or aspartate), (c) a small, semipolar, neutral amino acid (Ala, Ser, Cys, Thr) (Arriza et al., 1993 ; Ogawa et al., 1998 ), (d) both neutral and acidic amino acids or (e) most zwitterionic and dibasic amino acids (Reizer et al., 1994 ; Palacín et al., 1998 ; Zarbiv et al., 1998 ). The bacterial members are of about 450 (420–491) aminoacyl residues in length while the mammalian proteins are of about 550 (503–574) residues. These proteins possess between 10 and 12 putative TMSs (Slotboom et al., 1999 ).

All of the bacterial proteins cluster together on the phylogenetic tree as do the mammalian proteins (Saier et al., 1999a ). The mammalian permeases that transport neutral amino acids cluster separately from those that are specific for acidic amino acids. Among the mammalian proteins are neuronal excitatory amino acid neurotransmitter permeases. One of these, the Glt-1 L-glutamate/L-aspartate/D-aspartate transporter co-transports the neurotransmitter with 3 Na⁺ and 1 H⁺ and countertransports it against 1 K⁺ (Clark & Amara, 1993 ).

The MC (mitochondrial carrier) family (TC 2.A.29)
Permease protein subunits of the MC family possess six TMSs and exist in the mitochondrial membrane as homodimers (Aquila et al., 1987 ; Walker & Runswick, 1993 ; Palmieri, 1994 ; Palmieri et al., 1997 , 1999 ; Tzagoloff et al., 1996 ; Echtay et al., 1998 ; Feirmonte et al., 1998 ; Schroers et al., 1998 ). The proteins are of fairly uniform size, about 300 residues (Kuan & Saier, 1993 ; Indiveri et al., 1997 ). They arose by tandem intragenic triplication such that a genetic element encoding two TMSs gave rise to one encoding six TMSs (Saraste & Walker, 1982 ; Walker & Runswick, 1993 ). This event may have occurred when mitochondria first developed their specialized but permanent organellar functions within eukaryotic cells from endosymbiotic bacteria (Kuan & Saier, 1993 ; Indiveri et al., 1997 ). Members of the MC family are found exclusively in eukaryotic organelles although they are nuclearly encoded. Most are found in mitochondria, but some are found in peroxisomes of animals and in amyloplasts of plants (McCammon et al., 1990 ; Sullivan et al., 1991 ; see Kuan & Saier, 1993 for a review). Many of them preferentially catalyse the exchange of one solute for another (antiport); but two of them, the aspartate/glutamate exchanger and the ADP/ATP exchanger can function as anion-selective channels after chemical treatment with thiol reagents (Dierks et al., 1990a , b ). Thirty-four paralogues of the MC family are encoded within the genome of Saccharomyces cerevisiae, and 35 are encoded within the Caenorhabditis elegans genome (Paulsen et al., 1998b ; I. T. Paulsen, S. R. Goldman, W. S. Barnes and M. H. Saier, Jr, unpublished).

The AEC (auxin efflux carrier) family (TC 2.A.69)
Plants possess tissue-specific, PMF-driven, polarly localized cellular auxin efflux systems (Gälweiler et al., 1998 ; Luschnig et al., 1998 ). These carriers are saturable, auxin specific and localized to the basal ends of auxin-transport-competent cells. They may be found in various plant tissues including vascular tissues and roots. They are responsible for the polar (downwards) transport of auxins from the leaves to the roots and function in gravitropism. A single plant such as Arabidopsis thaliana may possess over six genes encoding such systems. Two isoforms, one in vascular tissue (PIN1) and one in roots (REH1) have been functionally characterized as has a homologue from Oryza sativa. These plant proteins are 600–700 aminoacyl residues long and exhibit 8–12 TMSs.

Homologues of the AEC family are found in bacteria (Escherichia coli, Klebsiella pneumoniae, Synechocystis, Aquifex aeolicus, Bacillus subtilis and Rickettsia prowazekii) as well as in archaea (Methanococcus jannaschii and Methanobacterium thermoautotrophicum). The Klebsiella pneumoniae homologue (MdcF, 319 aa) has been suggested to function in malonate uptake (Hoenke et al., 1997 ; Dimroth & Hilbi, 1997 ). The bacterial proteins are generally of 300–400 aa in length.

Yeasts also possess homologues of the AEC family. Saccharomyces cerevisiae has three functionally uncharacterized AEC family members (YL52, spP54072, 64·0 kDa; YNJ5, spP53930, 71·2 kDa; and YB8B, spP38355, 47·5 kDa), and Schizosaccharomyces pombe also has at least one sequenced homologue. It is thus clear that members of the AEC family are widespread, being found in bacteria, archaea, fungi and plants. Caenorhabditis elegans, however, appears to lack identifiable homologues of the AEC family. Based on PSI-BLAST results, the AEC family may be distantly related to the bile acid:Na⁺ symporter (BASS) family (TC 2.28), which is represented in animals.

	Families of secondary transporters specific for amines, amides and polyamines

TOP Overview Background Characteristics of transporters... Most amino acid transporters... ABC (ATP-binding cassette) -type... Secondary carriers Secondary amino acid transporter... Amino acid transporter families... Families of secondary... Families of channel-forming... Families of peptide transporters Protein-transport systems Conclusion REFERENCES

 
In addition to the ABC superfamily, which includes members that transport various amines, amides and polyamines, several of the amino acid transporter families described above have been shown to include members that are capable of transporting these compounds. These families include the MFS, APC, BCCT, AAAP, SSS, NSS, MC and AEC families (see Table 2). Additionally, the CAAT family has been implicated in amine transport (Ferguson & Krzycki, 1997 ). Most of these families are described in Table 4, but the BCCT family and the CAAT family, being prokaryote-specific, are described in Table 3. There are no families of secondary carriers in which all members transport simple amines and/or amides exclusively, but not amino acids. Thus, none of the families listed above needs be described here. The fact that most families of permeases which include members that transport amino acids and their derivatives are exclusive for these compounds and do not include members that transport other classes of compounds (i.e. sugars, nucleobases and their derivatives, vitamins, etc.) emphasizes the value of the phylogenetic approach to the functional characterization of recognized permease homologues.

	Families of channel-forming proteins capable of transporting amines and amides

TOP Overview Background Characteristics of transporters... Most amino acid transporters... ABC (ATP-binding cassette) -type... Secondary carriers Secondary amino acid transporter... Amino acid transporter families... Families of secondary... Families of channel-forming... Families of peptide transporters Protein-transport systems Conclusion REFERENCES

 
Three families of channel-forming proteins appear to be capable of transporting simple amines and/or amides. These families are listed in Table 5 and are described below.

MIP family proteins are believed to form aqueous pores that selectively allow passive transport of their solute(s) across the membrane with minimal apparent recognition (Chrispeels & Maurel, 1994 ; Shukla & Chrispeels, 1998 ). Aquaporins selectively transport water (but not glycerol), while glycerol facilitators selectively transport glycerol but not water (Maurel et al., 1993 ). Glycerol facilitators function as solute nonspecific channels, and may transport glycerol, dihydroxyacetone, propanediol, urea and other small straight-chain neutral molecules in physiologically important processes (Heller et al., 1980 ; Maurel et al., 1994 ). A few members of the family, including the yeast FPS protein (TC 1.A.8.5.1), transport both water and glycerol. Reports of MIP family proteins transporting ions may or may not be physiologically significant. However, demonstration of the involvement of the cyanobacterial channel protein (TC 1.A.8.4.1) in copper homeostasis suggests that it may transport Cu²⁺ (Kashiwagi et al., 1995 ).

The physiological functions of many MIP family proteins are unknown. They probably consist of homodimers (GlpF of Escherichia coli; TC 1.A.8.1.1) or tetramers (MIP of Bos taurus; TC 1.A.8.8.1). Each subunit spans the membrane six times as putative -helices and arose from a three-spanner-encoding genetic element by a tandem, intragenic duplication event (Reizer et al., 1993b ). Consequently, the two halves of the protein are in opposite orientations in the membrane, an unusual feature of a transport protein.

The urea transporter (UT) family (TC 1.A.44)
Members of the UT family are found only in mammals and amphibians (Olives et al., 1994 ; Couriaud et al., 1998 ). In a single species (i.e. rat or human) there may be at least three isoforms. One of the human UT family members is the Kidd (JK) blood group glycoprotein (Lucien et al., 1998 ). Most of these proteins vary in size from 380–400 residues and exhibit 10 putative TMSs, but one of them, a rat urea transporter (gbU77971), is reported to be 929 residues long (Shayakul et al., 1996 ). At least one of these proteins (UT3) can transport water as well as urea (Yang & Verkman, 1998 ). A channel-type mechanism is probable. Two members of this family, UT1 and UT2, may be derived from a single gene by alternative splicing.

The PLM (phospholemman) family (TC 1.A.27)
The PLM family includes mammalian phospholemmans of 8–10 kDa size (Chen et al., 1997 ; Foskett, 1998 ; Kirk & Strange, 1998 ). The human, rat and dog proteins have been sequenced and characterized. They span the membrane once with their N termini outside. These proteins induce a hyperpolarization-activated chloride current in Xenopus oocytes. They are found in muscle and many body tissues and are targets of protein kinases A and C. Other possible members include the chloride-conductance inducer protein, Mat8, and the Na⁺/K⁺ ATPase-subunit ‘proteolipid’ (TC 3.A.3). These proteins are smaller, but they exhibit the same orientation in the membrane.

PLM forms anion-selective channels when reconstituted in planar lipid bilayers. These channels display a linear current–voltage relationship, have a unitary conductance and are open most of the time at voltages between -70 and +70 mV. The PLM channel is permeable to both organic and inorganic anions including chloride, taurine, lactate, glutamate, isethionate and gluconate (Kirk & Strange, 1998 ).

	Families of peptide transporters

TOP Overview Background Characteristics of transporters... Most amino acid transporters...