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Keywords:

  • drug resistance;
  • metabolites;
  • nucleotide-sugars;
  • nutrients;
  • transport

Abstract

  1. Top of page
  2. Abstract
  3. Computer methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Previous work defined several families of secondary active transporters, including the prokaryotic small multidrug resistance (SMR) and rhamnose transporter (RhaT) families as well as the eukaryotic organellar triose phosphate transporter (TPT) and nucleotide-sugar transporter (NST) families. We show that these families as well as several other previously unrecognized families of established or putative secondary active transporters comprise a large ubiquitous superfamily found in bacteria, archaea and eukaryotes. We have designated it the drug/metabolite transporter (DMT) superfamily (transporter classification number 2.A.7) and have shown that it consists of 14 phylogenetic families, five of which include no functionally well-characterized members. The largest family in the DMT superfamily, the drug/metabolite exporter (DME) family, consists of over 100 sequenced members, several of which have been implicated in metabolite export. Each DMT family consists of proteins with a distinctive topology: four, five, nine or 10 putative transmembrane α helical spanners (TMSs) per polypeptide chain. The five TMS proteins include an N-terminal TMS lacking the four TMS proteins. The full-length proteins of 10 putative TMSs apparently arose by intragenic duplication of an element encoding a primordial five-TMS polypeptide. Sequenced members of the 14 families are tabulated and phylogenetic trees for all the families are presented. Sequence and topological analyses allow structural and functional predictions.

Abbreviations
MDR

multidrug resistance

ABC

ATP-binding cassette

MF

major facilitator

MFS

major facilitor superfamily

RND

resistance-nodulation-division

SMR

small multidrug resistance

MATE

multi-antimicrobial extrusion

DMT

drug/metabolite transporter

TMS

transmembrane α helical spanner

BAT

bacterial/archaeal transporter

DME

drug/metabolite exporter

GRP

glucose/ribose porter

RhaT

l-rhamnose transporter

CEO

Caenrhabditis elegans ORF

TPT

triose phosphate transporter

POP

plant organocation permease

NST

nucleotide-sugar transporter

UAA, UDP-N-acetylglucosamine 

UMP antiporter

UGA, UDP-galactose 

UMP antiporter

CSA, CMP-sialate 

CMP antiporter

GMA, GDP-mannose 

GMP antiporter

TC

transporter classification

Multidrug and drug-specific efflux systems are responsible for clinically significant resistance to chemotherapeutic agents in pathogenic bacteria, fungi, parasites and in human cancer cells (reviewed in [1,2]). Over 90% of the effort aimed at understanding multidrug resistance (MDR) has dealt with members of the major facilitator (MF) and ATP-binding cassette (ABC) superfamilies. The ABC superfamily includes the ATP-dependent multidrug efflux proteins such as P-glycoprotein and MRP, responsible for resistance of human tumor cells to anticancer chemotherapeutic agents, as well as PfMDR1 associated with chloroquine resistance in the malaria parasite Plasmodium falciparum[3]. Twenty-nine such ABC transporters are found in the completely sequenced genome of Saccharomyces cerevisiae[4–6]. A few functional and structural homologs of ABC-type MDR pumps have been identified in bacteria [7]. The MF superfamily includes protonmotive force-dependent secondary-drug efflux pumps in pathogenic micro-organisms such as QacA and NorA of Staphylococcus aureus and CaMDR1 of Candida albicans [1]. More than two dozen members of each of the MF and ABC superfamilies have been functionally characterized as MDR pumps (see our web site http://www-biology.ucsd.edu/~msaier/transport/ for details about these and other superfamilies).

Three remaining families of recognized bacterial MDR efflux pumps are the resistance-nodulation-division (RND), the small multidrug resistance (SMR) and the multi-antimicrobial extrusion (MATE) families. Whereas some of these MDR pumps may have evolved specifically for the purpose of expelling endogenously synthesized or exogenously derived toxic substances (e.g. transporters associated with Streptomyces antibiotic-biosynthetic operons) [8], others may transport drugs opportunistically (e.g. the Bacillus subtilis Blt drug pump which also expels polyamines) [9]. Thus, there may be overlap between drug-efflux and metabolite-efflux pumps. Nevertheless, each of the aforementioned superfamilies consists of members that have common structural and mechanistic features not shared by members of the other superfamilies. In this review, we define a novel ubiquitous superfamily that includes the SMR family but is unrelated to the other recognized drug-efflux superfamilies (ABC, MF, RND and MATE).

Nutrient-uptake systems in bacteria are usually energy coupled and therefore essentially unidirectional. Although this is also true of many eukaryotic uptake systems, higher organisms including animals often take up nutrients via energy-independent facilitated diffusion systems that catalyze fully reversible transport reactions. Consequently, whereas some eukaryotes use the same pathways for uptake and efflux, this is seldom true for bacteria [10,11]. Bacterial efflux systems for sugars [12,13], amino acids [14,15], and end products of metabolism [16–18] have been described. In animal cells, no such energized systems for efflux of sugars or amino acids have as yet been detected, and the few efflux systems for end products of metabolism that have been identified are very similar to, or even identical with, those catalyzing uptake (see, for example [19,20]).

Of the few examples of sugar/amino acid/metabolite-efflux pumps currently known, almost all have been described only within the last couple of years. Further, except for the work of Krämer [10,11], there was, until recently, little recognition of the physiological need for such systems, particularly for essential nutrient building blocks such as sugars and amino acids. Metabolite efflux therefore represents a poorly understood and little studied physiological phenomenon. Evidently, the toxic effects of high concentrations of sugars, amino acids, other metabolites, and their detrimental analogs, have provided evolutionary pressure for the acquisition of these efflux systems. To what extent they will prove to be important, and the number of exporter families that will provide these functions, remain to be determined. Our phylogenetic analyses of transport systems have led us to suggest that numerous prokaryotic permease families are concerned exclusively with solute export.

The sugar-efflux transporter (SET) family [transporter classification (TC) no. 2.1.20) is a group of recently described permeases within the MFS[12,13]. Members of this family include proteins from a wide variety of Gram-negative and Gram-positive bacteria, although functional data are available only for two closely related Escherichia coli members (see [21] for a review). The two characterized permeases, SetA and SetB, both mediate efflux of glucose and lactose, and SetA, but not SetB, also mediates efflux of isopropyl thio-β-d-galactoside, galactose and certain sugar-containing antibiotics. SetA-mediated efflux is inhibited by a large variety of d sugars and l sugars as well as glycosides of α-anomeric and β-anomeric configurations, suggesting broad specificity. Although not established, a proton : sugar antiport mechanism was inferred.

Bost et al. [22] and Caroléet al. [23] have both identified the E. coli protein, YdeA, a member of the drug : H+ antiporter family (DHA2) (TC no. 2.1.2) of the major facilitator superfamily (MFS), as a sugar-efflux pump that accommodates both l-arabinose and isopropyl β-d-thiogalactopyranoside. This system will undoubtedly prove to be of broad specificity, as has been documented for SetA.

Krämer, Eggeling and their coworkers described an efflux pump for l-lysine and l-arginine, the so-called LysE protein of Corynebacterium glutamicum[14,15,24]. Recently, two distantly related members of this family in E. coli, RhtB and RhtC, were suggested to mediate efflux of homoserine and threonine, respectively, based primarily on growth inhibition studies [25,26]. E. coli alone has at least six paralogs of this family, all of which may prove to be amino-acid-efflux systems [27]. The LysE superfamily (TC families 2.75–2.77) may comprise a superfamily of permeases devoted solely to efflux.

These examples may represent just the tip of the iceberg. We predict that many members of these families, as well as many proteins in new families, will prove to function in the efflux of metabolites, macromolecules and hydrophobic substances. Most of these families are also expected to be prokaryote specific.

In this article we describe a ubiquitous superfamily that includes members known to mediate (a) export of drugs, (b) nutrient uptake, (c) efflux of nutrients and metabolites, and (d) exchange of metabolites across intraorganellar membranes of eukaryotes. Most of the members of this superfamily belong to families for which few or no functional data are available. For the prokaryotic members, we expect that many of the proteins will prove to be pumps for drug or metabolite efflux. For the eukaryotic members, organellar metabolite or drug transport have been established or seem quite probable. We summarize published evidence for the functions of some of these proteins and suggest functions for some of the others. The work reported serves to characterize the interrelationships of members of this superfamily. It should provide a guide for the elucidation of functional characteristics for the diverse group of proteins that comprise the 14 families of the drug/metabolite transporter (DMT) superfamily.

Computer methods

  1. Top of page
  2. Abstract
  3. Computer methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Sequences of the proteins that comprise the 14 families within the DMT superfamily were obtained by an initial screening procedure involving recursive psiblast searches without iteration until all recognizable members had been retrieved from the NR databases (e value ≤ 10−4 as obtained with this program) [28]. This value suggests but does not establish homology as detailed in the second paragraph of this section (see also Table 3 for rigorous proof of homology). Subsequently, phylogenetic trees were constructed based on multiple alignments developed with the clustal x 1.8 [29] and tree[30] programs. Average hydropathy, similarity and amphipathicity plots were generated using the alignment based on the tree program, with a sliding window of 21 residues with hydrophobicity values as described by Kyte & Doolittle [31]. Amphipathicity plots were generated with an angle set at 100 ° as is appropriate for an α helix. The clustal and tree programs gave concurring results (see [32] for evaluation of these and other programs concerned with phylogenetic tree construction).

Table 3. Interfamilial binary comparisons for the 14 families of the DMT superfamily. The gap program was used to align the sequences with a gap opening penalty of 8, a gap extension penalty of 2 and 300 random shuffles.
Family Name gi number ResiduesTotal residues vs Family Name gi number ResiduesTotal ResiduesGap score SD
SMROrf1 Sty292142439–107111 DMEOrf1 Ssp100176291–15933716.3
SMRY4nH Rsp249671648–110117 DMEOrf1 Hin157389576–13730614.6
SMROrf1 Sco542002740–102106 BATOrf1 Mja282639762–13513710.2
BATOrf1 Mja282639740–135137 DMEOrf1 Spy 596092178–27427710.3
GRPGlcU Sxy22260011–288288 DMEOrf6 Pho32570191–28528512.1
CEOOrf1 Cel38773641–330330 DMEOrf1 Lhe38500471–2802809.4
DMEOrf1 Ssp100176290–159337 BATOrf1Aae298405766–13314313.6
DMEYcxE Bsu664809553–180287 RhaTRhaT Eco 13252836–16034410.1
DMEPago Sty60936441–304304 P-DMEOrf1 ath6957732120–43243210.1
TPTCptP Zma1352200101–409409 DMELicB1 Hin  971681–30530518
RarDY680 Hin28334921–298298 DMEOrf1 Pab54575101–29529510.3
UGACGI-19 Hsa46806771–382382 UAAOrf1 Cel74978611–31831818.5
TPTOrf3 Ath336751551–410410 UGAOrf1 Rno21363481–32232217.6
DMEYcaY Ckl 7313391–311311 POPOrf21 Ath7487848751–112811309
DMELicB1 Hin  971681–305305 TPTOrf1 Ath3983125100–41041013.2
TPTCpt2 Bol1706110101–402402 GMAOrf4 Ath74863281–29629616
DMEYoaV Bsu61372611–292292 CSACmsT Cgr24992251–33633611.3

Family assignments were based on the phylogenetic results and on the statistical analyses obtained with the gap program [33]. Thus, our standard criterion for establishing homology between two proteins is 9SDs, when two comparable regions of more than 60 residues (the size of a typical protein domain) are compared using the gap program, with 500 random shuffles, a gap opening penalty of 8, and a gap extension penalty of 2 [33], as outlined and rationalized by Saier [34].

The main features of the 14 families are presented in Table 1, the protein members of the 14 families are presented in Table 2, and the gap scores for representative interfamilial sequence comparisons are presented in Table 3. Comparison scores for members of a single family within the DMT superfamily always exceeded 15 SDs. Phylogenetic analyses of all members of these 14 families are presented in the text, and inclusion of outliers (members of the other 13 families) always revealed that they were more distantly related to the member of a family than the members of that family were to each other. Thus, family assignment was always based on phylogeny, and comparison scores using the gap program established distant phylogenetic relationships.

Table 1. The 14 established families in the DMT superfamily.
Family TC no. Family name Abbr.No. of membersaSource organismsbSize range (no. residues)No. putative TMSscWell-characterized examplesd
  • a

    Number of sequenced members in the current databases.

  • b

    b B, bacteria; Ar, archaea; E, eukaryotes; within the eukaryotic domain: Y, yeast; An, animals; Pl, plants.

  • c

    c Predicted topologies are based on average hydropathy plots for a subset of members from each of the families.

  • d

    See Table 2 for descriptions of these proteins.

2.A.7.14 TMS small multidrug resistanceSMR30B103–121 4EmrE Eco
2.A.7.25 TMS bacterial/archaeal transporterBAT6B, Ar137–143 5
2.A.7.310 TMS drug/metabolite exporterDME102B, Ar246–35310YdeD Eco
2.A.7.4Plant drug/metabolite exporterP-DME28Pl251–43210
2.A.7.5Glucose/ribose porterGRP6B280–29410RbsU Lsa
2.A.7.6l-rhamnose transporterRhaT2B34410RhaT Eco
2.A.7.7Chloramphenicol-sensitivity proteinRarD9B258–300 9 or 10
2.A.7.8C. elegans ORFCEO6An202–839 9 or 10
2.A.7.9Triose phosphate transporterTPT53An, Pl, Y246–520 6–9CptR Zma
2.A.7.10UDP-N-acetylglucosamine : UMP antiporterUAA7An, Y316–35210Mnn2 Kla
2.A.7.11UDP-galactose : UMP antiporterUGA15An, Pl, Y322–465 9 or 10Orf1 Hsa
2.A.7.12CMP-sialate : CMP antiporterCSA24An, Y263–42410Orf2 Mmu
2.A.7.13GDP-mannose : GMP antiporterGMA11An, Pl, Y249–37210Gog5 Sce
2.A.7.14Plant organocation permeasePOP12Pl315–112810, 30Pup1 Ath
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Figure 2. List of protein members of all 14 DMT families.

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Figure 2. Continued.

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Results

  1. Top of page
  2. Abstract
  3. Computer methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The DMT superfamily

Table 1 presents a summary of the 14 families of the DMT superfamily. The TC numbers and the names and abbreviations for each of the families are presented in columns 1–3. The number of sequenced protein members in each of these families currently in the GenBank, PIR and SwissProt databases are tabulated in column 4. Column 5 lists the kingdoms from which members of these families have been identified. Columns 6 and 7, respectively, present the protein size in terms of the number of aminoacyl residues and the number of putative transmembrane α helical spanners (TMSs) per polypeptide chain, based on average hydropathy plots (see Fig. 13 below). Finally, a functionally characterized example, when available, is provided in column 8.

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Figure 13. Average hydropathy (solid lines) and average similarity (dotted lines) plots for eight of the 14 families of the DMT superfamily. The remaining families (not shown) exhibited apparent 8–10 TMS topologies. The families depicted are indicated according to the family abbreviation, and the family numbers (see Tables 1 and 2) are indicated in parentheses. The tree program was used to align the sequences, and all plots were based on these alignments.

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The families will be described in detail when their phylogeny is discussed, but their characteristics will be briefly mentioned in the next section. The complete list of protein members in all 14 families can be found in Table 2. Table 3 presents the statistical analyses supporting the conclusion that all 14 families are related. A comparison score of 9 SDs corresponds to a probability of less than 10−19 that the sequence similarity exhibited by the two proteins occurred by chance [34–37].

Brief description of the 14 families within the DMT superfamily

Family 1 (SMR) consists of small, hydrophobic bacterial proteins with just four TMSs per polypeptide chain. Functionally characterized members are cation-specific multidrug efflux pumps.

Family 2 [bacterial/archaeal transporter (BAT)] includes members that are derived from both archaea and bacteria. No member of this family of five putative TMS proteins is functionally characterized.

Families 3 and 4 [drug/metabolite exporter (DME) and plant drug/metabolite exporter (P-DME)] are proteins of 10 putative TMSs which clearly exhibit an internal repeat sequence that must have arisen by intragenic duplication. They are consequently about twice as large as BAT family members. The DME family is the largest family in the DMT superfamily, having 102 currently sequenced prokaryotic members. Indirect evidence suggests that at least some of these proteins are efflux pumps for amino-acid metabolites and their toxic derivatives [38]. The P-DME family, found exclusively in plants, is more closely related to the proteins of the DME family than to any other DMT superfamily member. It is distinguished from the latter family primarily on the basis of origin and protein size. None of the P-DME family proteins is functionally characterized.

Families 5 and 6 [glucose/ribose porter (GRP) and l-rhamnose transporter (RhaT), respectively] include bacterial sugar-uptake permeases. Functionally characterized members of these two small families transport d-glucose, d-ribose and l-rhamnose.

The seventh family (RarD), also of bacterial origin, includes one member, RarD, which is described in the database as a chloramphenicol-sensitivity protein. No published description is available for this protein.

Six of the first seven families are prokaryotic specific, and the remaining seven families are found exclusively in eukaryotes. Thus, no family includes both prokaryotic and eukaryotic members. Family 8 [Caenrhabditis elegans ORF (CEO)] includes members found exclusively in C. elegans. None of these proteins is functionally characterized.

The triose phosphate transporter (TPT) family (family 9) is the largest of the eukaryotic families. It includes the well-characterized triose phosphate and sugar phosphate permeases of chloroplasts. However, it also includes functionally uncharacterized members found in yeast and animals.

Families 10–13 are all nucleotide-sugar transporters found in the endoplasmic reticular and Golgi membranes of eukaryotic cells. Each family includes members that are selective for a different nucleotide-sugar (UDP-N-acetylglucosamine, UDP-galactose, CMP-sialate or GDP-mannose), all of which function by a nucleotide antiport mechanism.

The last recognized family, the plant organocation permease (POP) family, with representation exclusively in plants, includes one functionally characterized member. This transporter appears to exhibit broad specificity for a wide range of structurally dissimilar organocations, including purines, phytohormones, alkaloids and drugs [39].

Phylogenetic analyses of the 14 families in the DMT superfamily

The SMR family.  SMR family pumps are prokaryotic transport systems consisting of homo-oligomeric or hetero-oligomeric structures [40–42]. The subunits of these systems are 100–120 aminoacyl residues in length and span the membrane as α helices four times (Table 1). Functionally characterized members of the SMR family catalyze efflux by a drug : H+ antiport mechanism in which the protonmotive force is used to drive expulsion of multiple drugs. The drugs transported are generally cationic [40,43].

The best-characterized member of the SMR family is probably EmrE of E. coli. Multidimensional NMR studies conducted with this protein have revealed its asymmetric, dimeric structure at 7-Å resolution [44,44a]. By using the technique of scanning cysteine accessibility, Mordoch and coworkers have provided evidence that the pathway by which the drug traverses the membrane is in a hydrophobic environment [45]. A glutamate at the active site is essential for high-affinity binding of cationic drugs [46,47,47a]. A topologically similar MDR pump has been identified in mammals [48]. However, this protein shows no detectable sequence or motif similarity to SMR family members and does not appear to be a member of the DMT superfamily.

In an earlier paper, we grouped proteins of the SMR family into two distinct clusters which we called the Smr and Sug clusters [42]. The Smr cluster included several drug-resistance pumps including Smr of S. aureus and EmrE of E. coli, both of which have been purified, reconstituted as drug : H+ antiporters, and extensively studied [42,49–52]. The Sug cluster included proteins identified as suppressors of GroEL mutations. These proteins have not been shown to function in drug efflux despite substantial effort in this direction. A transport function has not been identified for any of these Sug proteins.

B. subtilis has seven paralogs of the SMR family and E. coli has four. We have recently cloned all seven B. subtilis genes into expression vectors. None of these clones alone conferred on E. coli cells a drug-resistance phenotype. However, some of the cloned B. subtilis homologs confer increased resistance to crystal violet and certain other dyes when a pair of such proteins is present [40,41,41a]. Possibly the two proteins of each pair form a hetero-oligomer, or one functions as a chaperone protein to allow proper folding and insertion of the other.

The current phylogenetic tree for the SMR family is shown in Fig. 1. The Smr subfamily of the SMR family is shown at the top half of the tree. All of the functionally well-characterized drug-resistance pumps, including the putative heterodimeric EbrAB system of B. subtilis[41], are found in this sequence-diverse subfamily. The Sug subfamily segregates from the Smr subfamily and is shown in the lower half of the tree. The two SugE proteins of E. coli and Citrobacter freundii, both of which have been examined for drug resistance with negative results, cluster tightly together. Currently, they have no known function. The YkkC and YvdS proteins of B. subtilis cluster loosely together, and the YkkDa and YvdR proteins, although divergent in sequence, are adjacent to one another on the tree. It seems reasonable to suggest that the YkkCD pair functions in a capacity similar to that of YvdSR. The two Deinococcus radiodurans proteins, SugE1 and SugE2, also cluster together.

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Figure 1. Phylogenetic tree for the SMR family.

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The BAT family.  The BAT family is a small six-member family of five TMS proteins from a diverse group of organisms. The family consists of three bacterial and three archaeal members (Table 2). No organism encodes more than one of these proteins. They are all of similar size (137–143 amino acids). The phylogenetic tree for the proteins of this family shows phylogenetic distances that correspond roughly to those of the source organisms, suggesting that at least some of them may be orthologs of similar function. Thus, the proteins from Pyrococcus abyssi and Pyrococcus horikoshii cluster together, as do the two Gram-negative bacterial proteins. The two remaining proteins, one from Aquifex aeolicus and one from Methanococcus jannaschii, do not cluster. As the Pyrococcus proteins do not cluster even loosely with the Methanococcus protein, and as the A. aeolicus protein is as distant (or more distant) from the other bacterial proteins as from the archaeal proteins, horizontal transfer of genetic material may have occurred. It should be noted that, when the proteins of this early diverging bacterium were analyzed, no consistent phylogenetic picture emerged [53]. Thus, lateral transfer of genes between this hyperthermophile and other prokaryotes may have been a frequent occurrence.

The multiple alignment of BAT family protein sequences, on which Fig. 2 was based, revealed 10 fully conserved residues, all hydrophobic or neutral, and 22 positions where only conservative substitution occurs. Almost all of the latter were also hydrophobic in nature. The best-conserved portion of the alignment proved to be in the C-terminal region in which a stretch of 57 residues showed no gaps, five identities, and 12 conservative substitutions. This region was also well conserved in the SMR and DME families (see Fig. 14 and Fig. 15 below).

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Figure 2. Phylogenetic tree for the BAT family.

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Figure 14. Binary alignment of the first half of a DME family protein with its second half. This protein is Orf1 from A. aeolicus (gbAE000680). *, conservation at indicated position; :, conservative substitution; ., weaker conservative substitution. Coloured boxes represent the following types of amino acids: blue, hydrophobic/nonpolar; green, polar; pink, charged; orange, glycine; yellow, proline.

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Figure 15. Alignment of segments of SMR, BAT and DME family proteins. The C-terminal regions of these protein sequences were aligned. Orf1 of A. aeolicus, selected for alignment in Fig. 14, was the DME protein used. DME-1 refers to its first half, while DME-2 refers to its second half. (A) Alignment of a segment from only the first half of the DME protein with segments from the SMR and BAT family sequences. (B) Alignment of the corresponding sequences also with that for the second half of the DME family protein. The two remaining proteins selected for alignment were SMR [the Qac protein of S. aureus (gbU81980)] and BAT [Yeb6 of Pseudomonas dentrificans (spP29939)] family proteins. *, conservation at indicated position; :, conservative substitution; ., weaker conservative substitution. Coloured boxes represent the following types of amino acids: blue, hydrophobic/nonpolar; green, polar; pink, charged; orange, glycine; yellow, proline.

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The DME family. The DME family is a large family of integral membrane proteins ranging in size from 287 to 310 aminoacyl residues and exhibiting 10 putative α helical TMSs. These proteins are derived from phylogenetically divergent bacteria and archaea. In addition, B. subtilis, E. coli, Streptomyces coelicolor and Archaeoglobus fulgidus have multiple paralogs.

The proteins that make up the DME family evidently arose by an internal gene-duplication event. This conclusion is supported by the fact that the first halves of these proteins are homologous to the second halves (see Fig. 13 below). Several members of the DME family have been implicated in solute transport. Thus, the MttP protein of the archaeon, Methanosarcina barkeri, may transport methylamine [54]; MadN is encoded within the malonate-utilization operon of Malonomonas rubra and may be an acetate-efflux pump [55]; PecM is encoded within a locus of Erwinia chrysanthemi controlling pectinase, cellulase and blue pigment production and may export the pigments produced by gene products encoded in the pecM operon [56], and YdeD of E. coli has been implicated in efflux of metabolites of the cysteine pathway [38].

The phylogenetic tree for the DME family is shown in Fig. 3. Most of the proteins branch from points near the center of this unrooted tree. The exceptions are noteworthy: the genomes of two species of Pyrococcus, P. horikoshii and P. abyssi have been sequenced, and both contain six homologs in the DME family. Examination of the tree reveals that all six of the P. abyssi proteins pair with a P. horikoshii protein. They undoubtedly represent six orthologous pairs. We therefore predict with a high degree of confidence that members of each pair of these orthologs will prove to serve the same function in these two archaea. Because the branch lengths separating these six pairs of proteins are the same ± 50%, we can further conclude that the rates of sequence divergence have been similar for all six pairs of proteins.

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Figure 3. Phylogenetic tree for the DME family.

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Other examples of probable orthologs in bacteria include (a) the YicL proteins of E. coli and Salmonella typhimurium (top of the tree in Fig. 3), (b) the YigM proteins of E. coli and S. typhimurium (bottom left of Fig. 3), (c) the Orf1 proteins of Chlamydia trachomatis and Chlamydophila pneumoniae (right side of Fig. 3), (d) Orf3 of B. subtilis and Orf4 of S. coelicolor (lower right), (e) the so-called PecM protein of D. radiodurans and YbiF of E. coli (lower right), and (f) YcxC of B. subtilis and Orf1 of Bacillus licheniformis (lower left). PecM of E. chrysanthemi may be orthologous to Orf3 of S. coelicolor and PecM of Vogesella indigofera (lower right), but it is not orthologous to PecM of D. radiodurans. The designation of the D. radiodurans ORF as PecM represents a case of inappropriate annotation. If MadN of M. rubra is in fact an acetate-efflux system, the same may prove to be true of the YigM proteins of E. coli, S. typhimurium and Pseudomonas aeruginosa. It is interesting to note that of the five Haemophilus paralogs, only one (YbbE; upper right in Fig. 3) appears to have an E. coli ortholog. This is surprising in view of the fact that the H. influenzae genome is substantially smaller than the E. coli genome in spite of the fact that these two organisms are relatively closely related.

Some very close homologs can be found in the DME family, including the LicB and LicB1 proteins of H. influenzae (upper right) and Ytr1 and Orf1 from two different isolates of Buchnera aphidicola (upper left). The two B. aphidicola homologs appear to have diverged from each other shortly before Buchnera diverged from enteric bacteria, assuming that both PagO of S. typhimurium and/or Orf1 of Yersinia enterocolitica is/are orthologous to the Buchnera proteins.

The complete multiple alignment on which the tree shown in Fig. 3 was based revealed no fully conserved residues. However, several residue positions exhibited only conservative substitutions in most or all of the proteins. Therefore, it appears that the alignment is essentially correct.

The P-DME family. The P-DME family represents a large subset of the DME family. All of these proteins are derived from plants, and they cluster loosely together on a phylogenetic tree that includes all members of the DME and P-DME families. All of these proteins appear to have 10 TMSs. If this suggestion proves to be correct, then the two halves of these proteins will have opposite orientations in the membrane. Hydropathy plots suggest that many of the proteins that comprise families 2.A.7.3–2.A.7.14 exhibit 10 putative TMS proteins. The P-DME family includes 28 proteins, but 21 of these proteins are from a single organism, Arabidopsis thaliana, the plant for which the most genome sequence data are available.

No member of the P-DME family is functionally characterized, although one of these proteins, Nodulin 21 of Medicago truncatula, may be involved in bacterial nodulation. As revealed by the phylogenetic tree shown in Fig. 4, several of the proteins in the P-DME family branch from points near the center of the tree, but clustering patterns, particularly of A. thaliana paralogs, suggest that some of these proteins arose by recent gene-duplication events. Thus, Orf10 and Orf11 (right hand side of Fig. 4) and Orf5 and Orf17 (upper left) arose by recent gene-duplication events, but Orf22 and Orf4 diverged from Orfs 10 and 11 somewhat earlier, but later than from Orfs 23 and 24. The P-DME family therefore includes sequence similar and dissimilar proteins.

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Figure 4. Phylogenetic tree for the P-DME family.

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The multiple alignment for the P-DME family resembles that for the DME family in having no fully conserved residues but many conservative substitutions, particularly for the hydrophobic and/or semipolar residues (G, A, S, T and C). Visual examination of this alignment clearly suggests that the proteins had been correctly aligned.

The GRP family. The GRP family includes two functionally characterized members, a glucose-uptake permease of Staphylococcus xylosus[57,58], and a probable ribose-uptake permease of Lactobacillus sakei[59]. Both proteins probably function by H+ symport.

The GRP family includes just six sequenced members, all from Gram-positive bacteria, which vary in length between 277 and 288 aminoacyl residues. The phylogenetic tree (Fig. 5) suggests that the two Bacillus proteins are orthologs, but the same is probably not true for the two Lactobacillus proteins because they are quite divergent in sequence. The multiple alignment of these six proteins revealed a high degree of sequence similarity, with 36 fully conserved residues, many being hydrophilic.

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Figure 5. Phylogenetic tree for the GRP family.

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The RhaT family. The RhaT family contains only two proteins, the rhamnose : H+ symporters of E. coli and S. typhimurium, both of which have been functionally characterized. These RhaT proteins are both 344 amino acids long with 10 putative TMSs [60]. Because these proteins are very similar but have no close homologs, an alignment and a phylogenetic tree were not derived for this family.

The RarD family. The RarD protein of Ps. aeruginosa is indicated in the database entry describing its sequences as a chloramphenicol-sensitivity protein. No published data are available to provide functional information about any member of the family. Nine proteins comprise the family, and, except for a single homolog from B. subtilis, these proteins are all derived from Gram-negative bacteria. Also note that E. coli has two paralogs and H. influenzae has three.

The multiple alignment of this family reveals considerable sequence divergence, with only eight residues being fully conserved. The phylogenetic tree (Fig. 6) shows that the two E. coli homologs are very similar in sequence except for three amino-acid substitutions plus divergent N-termini. The three H. influenzae proteins are quite divergent in sequence, although Y223 and OrfD are closer in sequence to each other than to any other member of the family.

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Figure 6. Phylogenetic tree for the RarD family.

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The CEO family. Six proteins, all from C. elegans, and all of unknown function, comprise the CEO family. These proteins are reported to vary in length from 202 to 839 residues. Three of the proteins have about 350 residues each. Orf5 (202 residues) exhibits only five putative TMSs and may be a fragmentary sequence. The multiple alignment reveals a considerable degree of sequence similarity, with 11 fully conserved residues, most within a well-conserved stretch of 60 residues near the C-termini of all of these proteins except Yrr6, which exhibits both N-terminal and C-terminal extensions. The phylogenetic tree (not shown) revealed that all six proteins are about equally divergent in sequence.

The TPT family. Functionally characterized members of the TPT family are derived from the inner envelope membranes of chloroplasts and nongreen plastids of plants [61,62]. However, homologs are also present in yeast. S. cerevisiae has three functionally uncharacterized TPT paralogs encoded within its genome. Under normal physiological conditions, chloroplast TPTs mediate a strict antiport of substrates, frequently exchanging an organic three-carbon compound phosphate ester for inorganic phosphate (Pi) [63,64]. Normally, a triose phosphate, 3-phosphoglycerate, or another phosphorylated C3 compound made in the chloroplast during photosynthesis exits the organelle to the cytoplasm of the plant cell in exchange for Pi. Experiments with reconstituted translocators in artificial membranes indicate that transport can also occur by a channel-like uniport mechanism with up to 10-fold higher transport rates [65,66]. Channel opening may be induced by a large membrane potential and/or by high substrate concentrations. Nongreen plastid and chloroplast carriers, such as those from maize endosperm and root membranes, mediate transport of C3 compounds phosphorylated at carbon atom 2, particularly phosphoenolpyruvate, in exchange for Pi. These are phosphoenolpyruvate : Pi antiporters. Glucose 6-phosphate has also been shown to be a substrate of some plastid translocators [67,68].

Each TPT family protein consists of about 400–450 aminoacyl residues with six to nine putative TMSs [68–70] (see Fig. 13). The actual number has been proposed to be six TMSs for the plant proteins as well as for mitochondrial carriers (TC no. 2.A.29) and members of several other transporter families. However, proteins of the TPT family do not exhibit significant sequence similarity to the latter proteins, and there is no evidence for an internal repeat sequence. TPT proteins may exist as homodimers in the membrane [68].

As indicated in Tables 1 and 2, the TPT family is specific to eukaryotes; it includes over 50 sequenced members, making it the largest of the eukaryotic families. The multiple alignment of the TPT family reveals considerable sequence divergence. Only one residue is fully conserved, but several residue positions exhibit conservative substitutions. The phylogenetic tree (Fig. 7) reveals clustering of proteins according to both type of organism and function. Thus, seven of the 16 major branches contain plant proteins, five contain animal proteins, and four contain yeast proteins.

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Figure 7. Phylogenetic tree for the TPT family.

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All plant proteins outside of the three functionally defined clusters are from A. thaliana, and these proteins are highly divergent in sequence. It can be expected that the sequence-divergent proteins will prove to exhibit very different substrate specificities.

Families 10–13, the nucleotide-sugar transporter families. Nucleotide-sugar transporters (NSTs) are found in the Golgi apparatus and the endoplasmic reticulum of eukaryotic cells. Members of the family have been sequenced from yeast, protozoans and animals. Animals such as C. elegans possess many of these transporters. Humans have at least two closely related isoforms of the UDP-galactose:UMP exchange transporter.

NSTs generally appear to function by antiport mechanisms, exchanging a nucleotide-sugar for a nucleotide. Thus, CMP-sialic acid is exchanged for CMP [71,72], GDP-mannose is preferentially exchanged for GMP [73,74], and UDP-galactose and UDP-N-acetylglucosamine are exchanged for UMP (or possibly UDP) [75]. Other nucleotide-sugars (e.g. GDP-fucose, UDP-xylose, UDP-glucose, UDP-N-acetylgalactosamine) may also be transported in exchange for various nucleotides, but their transporters have not been molecularly characterized [73]. Each compound appears to be translocated by its own transport protein. Transport allows the compound, synthesized in the cytoplasm, to be exported to the lumen of the Golgi apparatus or the endoplasmic reticulum, where it is used for the synthesis of glycoproteins and glycolipids [73,76]. Comparable transport proteins exist for ATP that phosphorylates proteins, and phosphoadenosine phosphosulfate, which is used as a precursor for protein sulfation [73]. It is not known if these transport proteins are members of the DMT superfamily.

The sequenced NSTs are generally about 320–340 aminoacyl residues in length and exhibit 8–12 putative TMSs (Table 1 and Fig. 13 below). An eight-TMS model has been presented by Kawakita et al. [76] for the human UDP-galactose transporter 1.

The UAA family. The UDP-N-acetylglucosamine:UMP antiporter (UAA) family consists of four animal proteins, two from C. elegans and two from D. melanogaster, as well as three yeast proteins, one each from three different yeast species (Table 2). The multiple alignment of these proteins, with lengths ranging from 316 to 352 residues (Table 1), reveals considerable sequence conservation with 31 fully conserved residues. The highest degree of sequence identity occurs in a C-terminal region of 60 residues that is gap free and includes 15 of the 31 identities.

The phylogenetic tree (Fig. 8) shows the three yeast proteins clustering loosely together at the top of the tree and the four animal proteins at the bottom. The two Drosophila paralogs cluster loosely together.

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Figure 8. Phylogenetic tree for the UAA family.

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The UGA family. The 15 proteins of the UDP-galactose: UMP antiporter (UGA) family are derived from plants, animals and yeast. Three paralogs are, respectively, found in A. thaliana, C. elegans and D. melanogaster. The functionally characterized members of the family are from mammals (Table 2). Each of the two yeast species represented encodes only one member of the UGA family.

The multiple alignment of the proteins of the UGA family reveals three regions of fairly high sequence identity. A total of nine residues are fully conserved, and five of these are charged residues (E, K, K, D and R).

The phylogenetic tree for the UGA family (Fig. 9) shows very close clustering of the three characterized mammalian UDP-galactose transporters which cluster loosely with two homologs from D. melanogaster and C. elegans, respectively (top, right). A distinct cluster includes three proteins from the human, fly and worm (upper left). Two of the plant paralogs are closely related, and the third is very distant. Finally, the two yeast orthologs cluster loosely together.

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Figure 9. Phylogenetic tree for the UGA family.

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The CSA family. The CMP-sialate:CMP antiporter (CSA) family consists of two dozen sequenced proteins, three of which are CMP-sialate transporters from the human, mouse and cricket. They are very closely related in sequence. Other proteins of the CSA family are listed in the databases as UDP-N-acetylglucosamine transporters and UDP-galactose transporters (Table 2). These proteins exhibit a high degree of sequence divergence, as revealed by the multiple alignment, with only a single residue (tryptophan) being fully conserved in all 24 proteins. Several other residues near this tryptophan are well conserved, showing only conservative substitutions.

The phylogenetic tree (Fig. 10) shows tight clusters for each of the types of functionally characterized nucleotide-sugar transporters, the CMP-sialate (CSA), UDP-galactose (UGA) and UDP-N-acetylglucosamine (UAA) transporters (see Table 2). Many other sequence-divergent members of this family are expected to serve dissimilar functions.

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Figure 10. Phylogenetic tree for the CSA family.

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The GDP-mannose:GMP antiporter (GMA) family. A single protein, a GDP-mannose transporter, present in yeast Golgi membranes, confers upon this family its name. Eleven proteins comprise the family (Table 2). They are highly divergent, as only one residue (serine) is fully conserved. The one functionally characterized GDP-mannose transporter of S. cerevisiae clusters together with a yeast ortholog and a yeast paralog (Fig. 11). Except for one C. elegans protein that clusters loosely with a human homolog, all remaining proteins branch from points near the center of the tree (Fig. 11).

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Figure 11. Phylogenetic tree for the GMA family.

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The POP family. A single functionally characterized organocation transporter from A. thaliana is included within the POP family. This member of the POP family (AtPUP1) has been shown to transport adenine and cytosine with high affinity [39]. Evidence on energy coupling suggested an H+ symport mechanism. Purine derivatives (e.g. hypoxanthine), phytohormones (e.g. zeatin and kinetin) and alkaloids (e.g. caffeine) proved to be competitive inhibitors, suggesting that they may be transport substrates. Thus, AtPUP1 may be a broad-specificity organocation transporter. At least 15 members of this family have been sequenced from A. thaliana. Other family members have been reported to exhibit different affinities for nucleobases.

Although Pup1 may exhibit broad-substrate specificity, it appears to function primarily as a nucleobase-uptake porter. Most POP family members are about the same size, but one (Orf21) is about three times larger and exhibits about 30 TMSs instead of the usual 10. A phylogenetic tree (not shown) indicated that a recent triplication event probably gave rise to this unique protein sequence. Thus, the three repeat segments of this protein are more similar to each other than to any other homolog depicted in the tree. This is the only recognized example that we know of in which a transport protein has undergone a triplication event that gave rise to a large transporter homolog three times larger than the other family homologs.

The POP family multiple alignment revealed fairly good conservation, with 11 fully conserved residues. The clustal x program aligned the first hydrophobic domain of Orf21 with the other homologs. Most of the fully conserved residues are structural residues or hydroxyl amino acids (serine or tyrosine). The phylogenetic tree for the 12 A. thaliana proteins (Fig. 12) reveals a certain degree of clustering (i.e. Orfs 29, 30 and 31 cluster together as do Orfs 21, 22 and 23, and to a lesser degree Orf24). Although Pup1 and Orf26 cluster together, the other proteins are distant homologs of each other (Fig. 12).

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Figure 12. Phylogenetic tree for the POP family. The three repeat sequences of Orf21 (Orf21-1, -2, and -3) occur in a single tight cluster (data not shown).

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Topological predictions

Average hydropathy plots (dark lines) and average similarity plots (light lines) for eight of the 14 families within the DMT superfamily are presented in Fig. 13. The SMR family exhibits four peaks of hydrophobicity and the BAT family exhibits five, but most of the remaining families exhibit about 10. Spacing between peaks is distinctive; however, the spacing is not necessarily the same for the two halves of each of these families of aligned proteins. This is particularly noteworthy for the RarD and TPT families. While many of these proteins may have arisen by a primordial gene-duplication event, this event may have been obscured by extensive sequence divergence in some of the families.

Sequence comparisons of the SMR, BAT and DME families

Figure 14 shows an alignment of the first half of a member of the DME family with the second half. The large number of fully conserved residues as well as conservative substitutions establishes that the two halves of this protein are homologous to each other. The gap program [33] gave a comparison score of greater than 9 SDs, a value sufficient to establish homology. The proteins of the DME family thus evidently arose by an internal gene-duplication event. The primordial protein, like the BAT family proteins, presumably exhibited five TMSs, and the duplication event gave rise to a protein with 10 TMSs with the two halves in inverted orientation in the membrane. That is, each TMS has an inverted orientation compared with the same TMS in the other half; the order of the TMSs is not inverted (see Fig. 16).

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Figure 16. Proposed pathway for the evolutionary interconversion of three topologically dissimilar proteins derived from the SMR (four TMSs), BAT (five putative TMSs) and DME (10 putative TMSs) families within the DMT superfamily. The 10-TMS topology with opposite orientation in the membrane for the two halves of the protein has recently been demonstrated experimentally [77]. The scheme does not mean to imply known directionality.

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Figure 15A shows an alignment of a segment of an SMR family protein with the corresponding part of a BAT family protein and that of the first half of a DME family protein. The degree of sequence similarity shown clearly suggests homology. When the corresponding second half of the DME family protein was added, the alignment proved to be less impressive but still persuasive of homology. The alignments shown in Figs 14 and 15 and other considerations suggest that these proteins arose via a pathway presented in Fig. 16. We suggest that a four-TMS SMR family protein was the precursor, and that addition of a single TMS at the N-terminus gave rise to the five-TMS BAT family proteins. We cannot, however, prove directionality, so it is equally possible that loss of an N-terminal TMS from a primordial BAT family protein gave rise to the SMR-type proteins. An intragenic duplication event then gave rise to the presumed 10-TMS protein topology of the DME family proteins. As indicated in Fig. 16 and noted above, these two halves would be expected to exhibit opposite orientation in the membrane.

Discussion

  1. Top of page
  2. Abstract
  3. Computer methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In this paper, we have described a superfamily of topologically diverse transporters and have proposed a pathway for their origin. The SMR family proteins exhibit four established TMSs; the BAT family exhibits five putative TMSs, and the DME family proteins exhibit 10 putative TMSs. The putative 10-TMS topology for one member of the DME family (PecM of E. chrysanthemi) has recently been established [77]. These data therefore establish that the two halves of PecM are of opposite orientation in the membrane. The pathway proposed for the evolution of these proteins is:

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It is impossible to say whether four gave rise to five, or five gave rise to four, but it seems highly likely that five gave rise to 10 (Fig. 16). Other families within the DMT superfamily include proteins that exhibit as many as 10 TMSs, but possibly as few as six. We have not investigated the pathways that may have led to the loss of TMSs from the putative primordial permease protein, presumably of 10 TMSs, in some of the families within the DMT superfamily. However, equivalent evolutionary events have been documented for other families [78–81]. Detailed topological analyses and further sequence comparisons of DMT superfamily members may shed further light on this.

The functions and sources of the proteins included in the DMT superfamily are worthy of note. Six of the 14 families are exclusively prokaryotic and eight are exclusively eukaryotic. Thus, none of the families cross the prokaryotic/eukaryotic barrier. This fact argues against frequent horizontal transmission of genetic material between these two groups of organisms. However, members of the DME and P-DME families are clearly more closely related to each other than they are to other DMT superfamily members. Although no functional data are available for P-DME family proteins, we suggest that these proteins are chloroplast/plastid transporters derived from transporters in cyanobacteria, the chloroplast precursor bacteria. This is supported indirectly by the observation that TPT family members are found in chloroplasts and plant plastids, whereas other eukaryotic DMT superfamily proteins are apparently localized to intracellular organelles such as the endoplasmic reticulum and Golgi apparatus rather than to the cytoplasmic membrane of the eukaryotic cell. In eukaryotes, subcellular localization is sometimes (but not always) an evolutionarily conserved trait [37,82]. Thus, none of the mitochondrial carrier family proteins have so far been found in eukaryotic cell plasma membranes; they are apparently restricted to organelles such as mitochondria and peroxisomes [83]. In contrast, both human ENT1 and ENT2 transporters of the equilibrative nucleoside transporter family (TC no. 2.A.57) have been shown to be present in both cell surface membranes and organellar membranes including mitochondria, nuclear envelopes and lysosomes (see [82] for a summary of the evidence). Why eukaryotic subcellular localization should be a well-conserved evolutionary trait in some families but not others is not at present apparent.

This paper brings together a large amount of functional data for transporters characterized from diverse sources. Phylogenetic relationships between these proteins were not previously recognized. Because these proteins are evolutionarily related, they are not only expected to exhibit basic structural similarities, they should also exhibit fundamental mechanistic similarities. The phylogenetic data presented should therefore facilitate interpretation and consolidation of data obtained from studies of many DMT superfamily porters. We hope that biochemists and molecular biologists with primary interests in this diverse group of transporters will take advantage of the reported relationships, and use them to provide guidance for the design of experiments and the interpretation of results.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Computer methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We wish to thank Mary Beth Hiller and Jason Tchieu for their assistance in the preparation of this manuscript. Work in the authors' laboratory was supported by NIH grants 2R01 AI14176 from The National Institute of Allergy and Infectious Diseases and 9RO1 GM55434 from the National Institute of General Medical Sciences.

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  1. Top of page
  2. Abstract
  3. Computer methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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Footnotes
  1. *Note: these two authors contributed equally to the work.

  2. Note: a web page is available at http://www-biology.ucsd.edu/~msaier/transport/