INTRODUCTION

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"To know truly is to know by causes." Francis Bacon

"To me life consists simply in this, in the fluctuation between two poles, in the hither and thither between the two foundation pillars of the world." Herman Hesse

Transport systems serve the cell in numerous capacities (118-123). First, they allow entry of all essential nutrients into the cytoplasmic compartment and subsequently into organelles, allowing metabolism of exogenous sources of carbon, nitrogen, sulfur, and phosphorus. Second, they provide a means for the regulation of metabolite concentrations by catalyzing the excretion of end products of metabolic pathways from organelles and cells. Third, they mediate the active extrusion of drugs and other toxic substances from either the cytoplasm or the plasma membrane. Fourth, they mediate uptake and efflux of ionic species that must be maintained at concentrations that differ drastically from those in the external milieu. The maintenance of conditions conducive to life requires a membrane potential, requisite ion concentration gradients, and appropriate cytoplasmic concentrations of all essential trace minerals that participate as cofactors in metabolic processes. Such conditions are required for the generation of bioelectricity as well as for the maintenance of enzymatic activities. Fifth, transporters participate in the secretion of proteins, complex carbohydrates, and lipids into and beyond the cytoplasmic membrane, and these macromolecules serve a variety of biologically important roles in protection against environmental insult and predation, in communication with members of the same and different species, and in pathogenesis. Sixth, transport systems allow the transfer of nucleic acids across cell membranes, allowing genetic exchange between organisms and thereby promoting species diversification. Seventh, transporters facilitate the uptake and release of pheromones, alarmones, hormones, neurotransmitters, and a variety of other signaling molecules that allow a cell to participate in the biological experience of multicellularity. Finally, transport proteins allow living organisms to conduct biological warfare, secreting, for example, antibiotics, antiviral agents, antifungal agents, and toxins of humans and other animals that may confer upon the organism producing such an agent a selective advantage for survival purposes. Many of these toxins are themselves channel-forming proteins or peptides that serve a cell-disruptive transport function. Thus, from a functional standpoint, the importance of molecular transport to all facets of life cannot be overestimated.

The importance of transport processes to biological systems was recognized more than half a century ago (43, 82). Thanks largely to concerted efforts on the part of Jacques Monod and his coworkers at the Pasteur Institute in Paris, who studied the mechanism of action of the Escherichia coli lactose permease, the involvement of specific carrier proteins in transport became established (22, 113). Since these early studies, tremendous progress has been made in understanding the molecular bases of transport phenomena, and the E. coli lactose permease has frequently been at the forefront (45, 60, 143). Initially, transport processes were characterized from physiological standpoints using intact cells. Cell "ghosts" in which the cytoplasmic contents had been released by osmotic shock proved useful, particularly as applied to human red blood cells and later to bacteria. Work with such systems provided detailed kinetic descriptions of transport processes, and by analogy with chemical reactions catalyzed by enzymes, the proteinaceous nature of all types of permeases became firmly established (reviewed by Kaback [58]).

With the advent of gene-sequencing technologies, the primary structures of permeases first became available. Hydrophobicity analyses of these sequences revealed the strikingly hydrophobic nature of various types of integral membrane transporters (19, 68, 70, 95). Current multidisciplinary approaches are slowly yielding three-dimensional structural information about transport systems. However, since only a few such systems have yielded to X-ray crystallographic analyses (see, for example, references 26, 140, and 142 as well as Table 21 below), we still base our views of solute transport on molecular models that provide reasonable pictures of transport systems and the processes they catalyze without providing absolute assurance of accuracy (45, 59, 143).

It is well recognized that any two proteins that can be shown to be homologous (i.e., that exhibit sufficient primary and/or secondary structural similarity to establish that they arose from a common evolutionary ancestor) will in general prove to exhibit strikingly similar three-dimensional structures (32), although a few exceptions have been noted (127). Furthermore, the degree of tertiary structural similarity correlates well with the degree of primary structural similarity. For this reason, phylogenetic analyses allow application of modeling techniques to a large number of related proteins and additionally allow reliable extrapolation from one protein member of a family of known structure to others of unknown structure. Thus, once three-dimensional structural data are available for any one family member, these data can be applied to all other members within limits dictated by their degrees of sequence similarity. The same cannot be assumed for members of two independently evolving families or for any two proteins for which common descent has not been established.

Similar arguments apply to mechanistic considerations. Thus, the mechanism of solute transport is likely to be similar for all members of a permease family, and variations on a specific mechanistic theme will be greatest when the sequence divergence is greatest. By contrast, for members of any two independently evolving permease families, the transport mechanisms may be strikingly different. Knowledge of these considerations allows unified mechanistic deductive approaches to be correctly applied to the largest numbers of transport systems, even when evidence is obtained piecemeal from the study of different systems.

The capacity to deduce and extrapolate structural and mechanistic information illustrates the value of phylogenetic data. However, another benefit that may result from the study of molecular phylogeny is to allow an understanding of the mechanistic restrictions that were imposed upon an evolving family due to architectural constraints. Specific architectural features may allow one family to diversify in function with respect to substrate specificity, substrate affinity, velocity of transport, polarity of transport, and even mechanism of energy coupling. By contrast, the architectural constraints imposed on a second family may not allow functional diversification. Knowledge of the architectural constraints imposed on a permease family provides a clear clue as to the reliability of functional predictions for uncharacterized but related gene products revealed, for example, by genome sequencing. Conversely, the functional diversity of the members of a permease family must be assumed to reflect architectural constraints, and thus phylogenetic and functional analyses lead to architectural predictions.

Finally, phylogenetic analyses provide valuable information about the evolutionary process itself. One can sometimes glean clues regarding the time of appearance of a family, the organismal type in which the family arose, and the pathway taken for the emergence of the family during evolutionary history. Occasionally, it is also possible to ascertain whether or not two distinct families arose independently of each other.

Over the past decade, my laboratory has devoted considerable effort to the phylogenetic characterization of permease families (118-120). This work has led us to formulate a novel classification system superficially similar to that implemented years ago for enzymes by the Enzyme Commission. The transporter classification (TC) system has been reviewed and recommended for adoption by a panel of experts chaired by A. Kotyk of the International Union of Biochemistry and Molecular Biology (IUBMB). In contrast to the Enzyme Commission, which based its classification system solely on function, we have chosen to classify permeases on the basis of both function and phylogeny. In this review, I describe our proposal, point out some of its strengths, and emphasize its flexibility for the future inclusion of yet-to-be-discovered transporters. We hope that the TC classification system will prove to be as useful as the enzyme classification system. Earlier treatises concerning the TC system and transport protein evolution have appeared (121-123, 127).

A detailed description of the TC system can be found on our World Wide Web site (http://www-biology.ucsd.edu/~msaier/transport/). This site will be continuously updated as new relevant physiological, biochemical, genetic, biophysical, and sequence data become available. Thanks to the participation of Andrei Lupas and the SmithKline-Beecham bioinformatics group (5), the TC system is being automated so that new sequences will automatically appear in multiple alignments and phylogenetic trees with minimal human intervention. The system will also provide a user-friendly search tool, called TransBase, so that the TC system can be readily accessed by keyword, TC number, gene name, protein name, sequence, and sequence motif. These advances will render the TC system increasingly accessible to the entire scientific community worldwide. In return, members of the scientific community are strongly encouraged to communicate novel findings and corrections to me by E-mail, phone, fax, or snail mail.

Almost all transmembrane transport processes are mediated by integral membrane proteins, sometimes functioning in conjunction with extracytoplasmic receptors or receptor domains as well as with cytoplasmic energy-coupling and regulatory proteins or protein domains (51, 112, 130, 139). Each such complex of these proteins and/or protein domains is referred to as a transport system, transporter, porter, permease system, or permease. These are all equivalent terms that are in general use by members of the transport community. A permease (porter) is a protein or protein complex that catalyzes a vectorial reaction, irrespective of whether or not it also catalyzes a chemical or electron transfer reaction that drives the vectorial process. Thus, many transport systems can be thought of as catalytic proteins or protein complexes analogous to enzymes or enzyme complexes. By definition, transporters facilitate vectorial rather than, or in addition to, chemical reactions. The preferred terms for these transport systems are transporters or porter.

Permease-mediated transport can occur by any one of three distinct but related processes. First and simplest is facilitated, equilibrative, or protein-mediated diffusion, a process that is not coupled to metabolic energy and therefore cannot give rise to concentration gradients of the transported substrate across the membrane. Two primary modes of facilitated transport have been recognized in biological systems: channel type and carrier type (Fig. 1). In channel-type facilitated diffusion, the solute passes in a diffusion-limiting process from one side of the membrane to the other via a channel or pore that is lined by appropriately hydrophilic (for hydrophilic substrates), hydrophobic (for hydrophobic substrates), or amphipathic (for amphipathic substrates) amino acyl residue moieties of the constituent protein(s). The structures of several such channel proteins have now been examined and elucidated by X-ray crystallographic techniques (see below). In carrier-type facilitated diffusion, some part of the transporter is classically presumed to pass through the membrane together with the substrate (143, 151). Whether or not this presumption is correct is not known, as no classical carrier has yet yielded to the analytical tools of the X-ray crystallographer.

View larger version (16K): [in this window] [in a new window]   FIG. 1.   Scheme illustrating the currently recognized primary types of transporters found in nature. These proteins are initially divided into channels and carriers. Channels are subdivided into -helical protein channels, -barrel protein porins (mostly in the outer membranes of gram-negative bacteria and eukaryotic organelles), toxin channels, and peptide channels. Carriers are subdivided into primary active carriers, secondary active carriers (including uniporters), and group translocators that modify their substrates during transport. Primary sources of chemical energy that can be coupled to transport include pyrophosphate bond (i.e., ATP) hydrolysis, decarboxylation, and methyl transfer. Oxidation-reduction reactions, light absorption, and mechanical devices can also be coupled to transport (see text). Secondary active transport is driven by ion and other solute (electro)chemical gradients created by primary active transport systems. The only well-established group-translocating system found in nature is the bacterial phosphoenolpyruvate:sugar PTS, which phosphorylates its sugar substrates during transport.

Carriers usually exhibit rates of transport that are several orders of magnitude lower than those of channels. Moreover, in contrast to most channels, they exhibit stereospecific substrate specificities. Although both channels and carriers may exhibit the phenomenon of saturation kinetics, this is a more common characteristic of carriers. Very few carriers have been shown to be capable of functioning by a channel-type mechanism, and the few that exhibit this capacity generally do so only after the protein has been modified, either by covalent or noncovalent ligand binding or by imposition of a large membrane potential. Moreover, while most channels are oligomeric complexes, many carriers can function as monomeric proteins. These observations led to the suggestion that channels and carriers are fundamentally, not superficially, different.

If energy expenditure is coupled to transmembrane solute translocation, then a system catalyzing facilitated diffusion can become an active transporter. Such a system is considered to be a primary active transporter if a primary source of energy (i.e., a chemical reaction, light absorption, or electron flow) is coupled to the process. It is considered to be a secondary active transporter if a secondary source of energy (i.e., an ion electrochemical gradient, termed the proton motive force [PMF] in the case of protons or the sodium motive force [SMF] in the case of sodium ions), generated at the expense of a primary energy source, is coupled to the process. The transport panel considered all of these terms to be acceptable.

Active transporters (or porters) can function by uniport, symport, or antiport. Uniporters (the preferred term), also called single-species transporters or facilitated diffusion carriers (the less-preferred terms), catalyze the transport of a single molecular species, and transport therefore occurs independently of the movement of other molecular species. Symporters (the preferred term), also classically called cotransporters, catalyze the transport of two or more molecular species in the same direction. The fact that a single point mutation in a symporter can convert a carrier into a uniporter (41, 62, 66, 75, 147) emphasizes the superficial distinction between these two types of carriers. Antiporters (the preferred term), also called countertransporters, exchange transporters, and exchangers, catalyze the exchange of one or more molecular species for another. Antiport processes can be subdivided into two categories: antiport of like molecules (i.e., solute-solute antiport) and antiport of unlike molecules (i.e., solute-cation antiport). Many uniporters and symporters also catalyze solute-solute antiport, sometimes at rates that are substantially greater than those of uniport or symport. Some carriers catalyze solute-solute antiport at rates that exceed those of uniport or symport by 10³- to 10⁵-fold, and uniport via these carriers is of little or no physiological consequence (110). Such systems are said to be obligatory antiporters or exchangers.

Accelerative solute-solute antiport or countertransport has long been considered to be a diagnostic characteristic of carriers. Early transport kineticists concluded that its demonstration eliminated the possibility that a transporter functions by a channel-type mechanism and suggested that clear boundaries exist between carriers and channels (79, 135). Subsequent observations that certain "carriers" could apparently be converted into "channels" by chemical treatment (16, 17, 28, 29, 56), by imposition of large membrane potentials (131, 132, 149), or by ligand binding (13) led many students of transport to consider these boundaries indistinct. Our in silico phylogenetic and protein structural analyses suggest that these examples may be special cases and tend to reemphasize the importance of the channel-versus-carrier distinction (123, 127).

A few carriers modify their substrates during transport. The best-characterized such system is the bacterial phosphotransferase system (PTS), which phosphorylates its sugar substrates using phosphoenolpyruvate as the phosphoryl donor. Sugars taken up from the external milieu via the PTS are thus released into the cytoplasm as sugar-phosphates. Any process in which the substrate is modified during transport is termed group translocation. Although originally proposed in different form by Peter Mitchell as a general mechanism, its occurrence appears to be highly restricted in nature.

As noted above, enzymes have long been classified in accordance with the directives and recommendations of the Enzyme Commission (31). The commission developed its directives decades ago, long before protein sequence data became available. Their system of classification was based solely on function. It was tacitly assumed that proteins of similar catalytic function would be closely related and that they therefore should be grouped together. We now know, however, that two different enzymes catalyzing exactly the same reaction sometimes exhibit completely different amino acid sequences and three-dimensional structures, function by entirely different mechanisms, and apparently evolved independently of each other, converging only with respect to the final reactions catalyzed. The enzyme classification system is thus limited in that it reflects only the reactions catalyzed by and the substrate specificities of the enzymes. It does not recognize the phylogenetic origins of these proteins and therefore does not reflect structural or mechanistic features.

As has been extensively documented, molecular phylogeny provides a reliable guide to protein structure and mechanism of action. It also provides an indication (albeit less definitive) of the specific process catalyzed and the substrate acted upon (127). Since the former characteristics are fundamental traits of a protein while the latter characteristics are more superficial traits, sometimes merely reflective of single amino acyl residue changes in a protein, it would be reasonable to suggest that as more and more sequence and phylogenetic data become available, these data should be used to provide the most reliable basis for protein classification. Since single amino acyl residue substitutions in permeases can give rise to different substrate-binding specificities (15, 23, 44, 94), these characteristics should be used in the final level of classification rather than in a primary level. We conclude that recognition of the evolutionary process provides a reliable guide to structure, mechanism, and function, although a few exceptions may exist (102, 127). If molecular phylogenetic studies can accurately retrace the evolutionary process, they should be used as a basis for any rational system of protein classification.

Some of the enzymes classified within the enzyme classification system are asymmetrically situated within an anisotropic, hydrophobic lipid membrane that separates two aqueous compartments. The resultant asymmetry allows these enzymes to catalyze vectorial as well as chemical modification reactions, as clearly enunciated decades ago by Peter Mitchell (83-85). Some of these integral membrane enzymes do, in fact, catalyze transmembrane transport of ions or other small solutes. However, most currently recognized solute permeases do not catalyze a chemical reaction and consequently are not included within the enzyme classification system. The comprehensive system of permease classification proposed here has the potential to encompass all types of transporters, both those that are currently recognized and those that are yet to be discovered.

Early studies revealed that transport proteins could be grouped into families based exclusively on the degrees of similarity observed for their amino acid sequences (118). The significance of family assignment remained questionable until the study of internal gene duplications that had occurred during the evolution of some of these families established that these families had arisen independently of each other, at different times in evolutionary history, following different routes (119). In this section I will evaluate and utilize both function and molecular phylogeny for the purpose of conceptualizing transport protein characterization and classification (see also reference 120).

According to the proposed classification system, now recommended by the transport nomenclature panel of the IUBMB, transporters are grouped on the basis of five criteria, and each of these criteria corresponds to one of the five entries within the TC number for a particular permease. Thus, a permease-specific TC number has five components, V, W, X, Y, and Z. V corresponds to the transporter class, while W corresponds to the subclass (see Table 1). X specifies the permease family (or superfamily), while Y represents the subfamily in a family (or the family in a superfamily) in which a particular permease is found. Finally, Z delineates the substrate or range of substrates transported as well as the polarity of transport (in or out). Any two transport proteins in the same subfamily of a permease family that transport the same substrate(s) using the same mechanism are given the same TC number, regardless of whether they are orthologs (i.e., arose in distinct organisms by speciation) or paralogs (i.e., arose within a single organism by gene duplication). The mode of regulation proves not to correlate with phylogeny and was probably superimposed on permeases late in the evolutionary process. Regulation is therefore not used as a basis for classification.

There are four recognized classes of transporters: channels, porters, primary active transporters, and group translocators (Table 1). Sequenced homologs of unknown function or mechanism and functionally characterized permeases for which sequence data are not available are included in a distinct class, class 9. Deficiencies in our knowledge will presumably be eliminated with time as more sequenced permeases become characterized biochemically and as sequences become available for the functionally but not molecularly characterized permeases. One additional class (class 8) is reserved for auxiliary transport proteins. It should be noted that each subclass of transporters has a two-digit TC number (V.W); each family has a three-digit TC number (V.W.X); each subfamily has a four-digit TC number (V.W.X.Y); and each permease type has a five-digit TC number (V.W.X.Y.Z).

As mentioned above, the primary level of classification in the TC system is based on mode of transport and energy-coupling source. The classes and subclasses of transporters currently recognized are listed below.

The current index of transport system families is presented in Table 2. There are more than 250 entries, each of which usually describes a single family. Some of these families are actually large superfamilies with more than a thousand currently sequenced members (e.g., the voltage-gated ion channel (VIC) family (TC 1.A.1) (88); the major facilitator superfamily (MFS) (TC 2.A.1) (96, 125), and the ATP-binding cassette (ABC) superfamily (TC 3.A.1) (130, 139)). Others are very small families with only one or a few currently sequenced members. Most families, however, are currently of intermediate sizes, with between 5 and 500 sequenced members.

All of the families included in Table 2 will undoubtedly expand with time, and new families will be identified. The availability of new protein sequences will occasionally allow two or more currently recognized families to be placed together under a single TC number. In a few cases, two families are already known for which some evidence is available suggesting that they are related, e.g., the monovalent cation:proton antiporter-1 (CPA1) and CPA2 families (TC 2.A.36 and 2.A.37), the nucleobase-cation symporter-1 (NCS1) and NCS2 families (TC 2.A.39 and 2.A.40), as well as the L-lysine exporter, resistance to homoserine/threonine, and cadmium resistance families (TC 2.A.75, 2.A.76, and 2.A.77, respectively) (124, 148). Such evidence is usually based on limited sequence and/or sequence motif similarities, common function, and/or similar protein size, topology, and structure. When "missing link" sequences or three-dimensional structural data become available so that proteins of two families can be unequivocally grouped together within a single family, the lower TC number will be adopted for all of the family members, and the higher TC number will be abandoned.

The rigorous criteria used to delimit a family have been defined previously (121, 122). Briefly, in order for two proteins to belong to the same family, they must exhibit a region of 60 residues or more, in comparable portions of the two proteins, that have a comparison score in excess of 9 standard deviations (27). At this value, the probability that the degree of sequence similarity observed for these two proteins occurred by chance is less than 10¹⁹ (25). It is considered that this degree of sequence similarity could not have arisen either by chance or by a convergent evolutionary process (32, 118). A minimum of 60 residues was arbitrarily selected because many protein domains in water-soluble proteins are of about this size.

The complete TC system is available on our web site (http://www-biology.ucsd.edu/~msaier/transport/), where the descriptions, primary references, and list of functionally characterized protein members of all families are provided. The whole-genome analysis data upon which this classification system was initially based are found on an included subportion of this web site, which was constructed under the guidance of Ian Paulsen (100, 101). This site will be updated continuously as new information becomes available. Anyone noting errors or incomplete listings is encouraged to contact me to provide the missing information and references.

As noted above, members of a transporter family generally utilize a single mode of transport and energy-coupling mechanism, thus justifying the use of these functional categories as the primary basis for classification. However, a few exceptions have been noted. First, the arsenite efflux permease (ArsAB; TC 3.A.4) of E. coli consists of two proteins, ArsA and ArsB. ArsB is an integral membrane protein that presumably provides the transport pathway for the extrusion of arsenite and antimonite (134, 153). ArsA is an ATPase that energizes ArsB-mediated transport. However, when ArsB alone is present, as in the case of the arsenical resistance pump of Staphylococcus aureus, transport is driven by the PMF (14). Expression of the E. coli arsB gene in the absence of the arsA gene similarly gives rise to PMF-driven transport. The presence or absence of the ArsA protein thus determines the mode of energy coupling.

The ArsB protein is a member of a large superfamily of ion transporters, the ion transporter superfamily, in which at least two families exhibit the unusual capacity of being able to incorporate auxiliary constituents that alter the transport characteristics of the carrier (107, 127). Such promiscuous use of energy is exceptionally rare and has been documented in only a very few instances. When such an effect is reported, we shall usually classify the permease in accordance with the more complicated energy-coupling mechanism (in this case, as an ATP-driven primary active transporter [class 3] rather than as a secondary carrier [class 2]). However, in this unique case, the TC nomenclature panel of the IUBMB has recommended that a second family describing the PMF-driven ArsB homologs be included in the TC system (TC 2.A.45), as many ArsB homologs function by ATP-independent, ArsA-independent mechanisms.

Examples of secondary carrier families in which promiscuous transport modes have been reported include the mitochondrial carrier family (TC 2.A.29) and the triose phosphate/nucleotide sugar transporter (TP-NST) family (TC 2.A.50). Proteins of both families are apparently restricted to eukaryotic organelles. Members of these families normally catalyze carrier-mediated substrate-substrate antiport and are therefore classified as secondary carriers. However, treatment of mitochondrial carrier family members with chemical reagents, such as N-ethylmaleimide or Ca²⁺ (16, 17, 28, 29, 56), or imposition of a large membrane potential () across a membrane into which a TP-NST family member has been incorporated (131, 132, 149), has been reported to convert these antiport-catalyzing carriers into anion-selective channels capable of functioning by uniport. Another secondary carrier that may be capable of exhibiting channel-like properties is the KefC protein of E. coli (13), which is a member of the CPA2 family (TC 2.A.37). "Tunneling" of ions and other solutes through carriers with little or no conformational change has been discussed (42). Again, the more complicated carrier-type mechanism, which appears to be relevant under most physiological conditions, provides the basis for classifying these proteins (i.e., as class 2 carriers rather than class 1 channels).

Table 3 summarizes some of the key characteristics of most of the transporter families that we have identified. Categories 1.D and 2.B (non-ribosomally synthesized channels and carriers, respectively), 8 (auxiliary transport proteins), and 9.B and 9.C (putative but uncharacterized transporters) have been omitted (compare Table 1 with Table 3). Table 3 provides the family TC numbers, the abbreviations of the families, and the substrates of transporters included within each family. Substrates that are common to one transporter are separated by commas, while substrates of different transporters within the family are separated by semicolons. Thus, in the major intrinsic protein (MIP) family (TC 1.A.8), aquaporins generally transport water but not organic compounds, while glycerol facilitators generally transport short, straight-chain polyols but not water. A few members of the family may transport both (see reference 97 for a review). A recent report has provided evidence that a member of the MIP family can accommodate anions (154), but this observation is of uncertain physiological significance.

Table 3 also includes the size ranges of the individual protein members of the families and the numbers of (putative) transmembrane -helical segments (TMSs) included within the permease polypeptide chains. All members of a family usually exhibit similar topological features, although several exceptions have been noted. When a homo- or heterooligomeric structure has been established for an intact permease, this fact is also indicated. Finally, the kingdoms in which members of the family have been identified, the approximate number of members that have been identified in each family, and representative examples of well-characterized members are also provided. The table is largely self-explanatory, but detailed information as well as primary and secondary references are provided on our web site and may be available in book form in the near future (Saier et al., unpublished data).

A table entitled Cross-Referencing Permeases by Accession Number is included in our web site. In this table, accession numbers for most of the proteins included in the TC system as of June 1999 are tabulated in alphabetical and numerical order. These may be of general utility to the student of transport, as their availability allows one to easily search all databases using the various BLAST search tools (3). Knowledge of a TC number allows one to quickly identify (i) the protein referred to, (ii) the transport system of which that protein is a constituent, (iii) the substrate specificity of that system, (iv) the family to which that permease belongs, (v) the mode of transport used, (vi) the energy-coupling mechanism used, and (vii) many of the characteristics of that permease family. Thus, cross-referencing by accession number is useful when trying to identify the family to which a newly sequenced protein belongs.

As noted above, one needs only to conduct a BLAST search, and all sufficiently similar homologs will be displayed. When the accession number of any one of these retrieved homologs is shown to correspond to one of the established members of a family, the family to which the newly sequenced protein belongs is immediately known. Furthermore, by identifying the proteins with the highest BLAST scores (smallest P values), one can immediately recognize the closest homologs. This information provides an indication of the most likely substrate specificity, energy-coupling mechanism, and physiological function of the newly sequenced permease protein. Cross-referencing of accession numbers and TC numbers therefore provides a simple and rapid approach to the initial characterization of a newly sequenced porter. I and my colleagues, working with Andrei Lupas (SmithKline-Beecham), are currently developing a search tool based on the TC system that will allow anyone to search the complete TC system using sequence, sequence motif, accession number, gene name, protein name, family name, etc. (A. Lupas et al., unpublished data).

In 1993 and again in 1996, Monica Riley presented an extensive tabulation of E. coli gene products (114, 115). To facilitate this endeavor, enzymes were classified based on the nature of the molecule(s) (substrates) acted on. In order to cross-reference transport systems based on substrate specificities, a basis for classifying potential substrates had to be devised. We have done so, creating a system that includes virtually all currently recognized transport substrates. This system of cross-referencing transporters is described here.

All known transport substrates have been classified into eight categories (Table 4). These categories are I, inorganic molecules; II, carbon compounds, III, amino acids and derivatives; IV, bases and derivatives; V, vitamins, cofactors, signaling molecules, and their precursors; VI, drugs, dyes, sterols, and toxic substances; VII, macromolecules; and VIII, miscellaneous compounds.

Most inorganic molecules (category I) are cationic or anionic. However, some channel proteins are nonselective, and aquaporins of the MIP family (TC 1.1) transport water selectively. The four defined subcategories for category I therefore include A, nonselective; B, water; C, cations; and D, anions. Inorganic compounds not falling into one of these subcategories are classified as others (subcategory E), and this subcategory can be subdivided in the future if desired.

Carbon compounds (category II) have similarly been grouped into four defined subcategories: A, sugars, polyols, and their derivatives; B, monocarboxylates; C, di- and tricarboxylates; and D, noncarboxylic organic anions (organophosphates, phosphonates, sulfonates, and sulfates). Subcategory E (others) encompasses all other carbon compounds.

Amino acids and their derivatives (category III) have been subdivided into A, amino acids and conjugates; B, amines, amides, and polyamines; C, peptides; D, other related organocations; and E, others. Bases and their derivatives (category IV) have been subcategorized into A, nucleobases; B, nucleosides; C, nucleotides; D, other related derivatives; and E, others. Vitamins, cofactors, and cofactor precursors (category V) have been subcategorized into A, vitamins and vitamin or cofactor precursors; B, enzyme and redox cofactors; C, siderophores and siderophore-iron complexes; D, signaling molecules; and E, others. Drugs, dyes, sterols, and toxics (category VI) have been subcategorized into A, multiple drugs and dyes; B, specific drugs; C, bile salts and conjugates; D, sterols and conjugates; and E, others. Category VII is devoted to macromolecules: A, complex carbohydrates; B, proteins; C, nucleic acids; D, lipids; and E, others. Finally, category VIII (miscellaneous) encompasses any transport substrate that does not fall into categories I to VII. So far no transport substrate has been relegated to category VIII, and very few of those in categories I to VII have fallen into the "other" category.

A few compounds belong to more than one category. For example, bile acids fall into both category II.B and category VI.C. Theoretically, oligosaccharides (e.g., lactose, raffinose, and maltooligosaccharides) could be classified either in II.A or in VII.A. We have elected to put oligosaccharides into category II.A and reserve category VII.A for larger molecules such as polysaccharides, teichoic acids, and lipooligosaccharides. Thus, category II.A generally refers to smaller carbohydrates normally taken up by cells for purposes of carbon catabolism, while category VII.A refers to structural carbohydrates that are synthesized by cells and exported intact.

Some permease systems transport a range of compounds that fall into more than one category. For example, a single ABC export system may catalyze efflux of multiple drugs (VI.A) and peptides (III.C), and it may also facilitate phospholipid flipping between the two bilayers of a membrane (VII.D). Such permeases are rare, but when they do occur, they will be included in all applicable categories.

As described above, Table 4 groups potential transport substrates according to structure and biological significance. This system of substrate classification has been used to cross-reference transport systems according to the types of substrates and the specific substrate(s) transported.

Table 5 presents the distribution of transporter types based on substrate specificity. In this table, permeases are categorized into four groups: -type channels, -type porins, primary carriers (regardless of the primary source of energy utilized and including PTS-type group translocators), and secondary carriers (including uniporters, symporters, and antiporters). Transporter types of unknown mode of transport or energy-coupling mechanism (categories 9.A and 9.B) were not included in Table 5.

-Type channel proteins (TC 1.A) generally either are nonselective (13 types) or function in the transport of inorganic ions (I.C and I.D; 18 families) or proteins (VII.B; 5 families). One type (aquaporins in the MIP family; TC 1.A.8) transports water, while another type of the same family (glycerol facilitators of the MIP family) transports straight-chain polyols and small organic molecules such as urea. Some MIP family proteins may transport both water and small, neutral organic molecules, but with the possible exception of a single MIP family member (154), none of the MIP family channel proteins have been shown to be selective for ions or larger molecules. Besides MIP family members, only two other recognized channel families include members that are specific for organic compounds. These families are the urea transporter family (TC 1.A.44) and the phospholemman (PLM) family (TC 1.A.27). The PLM family includes members that transport organic anions. Channel-forming toxins (TC 1.C) are generally nonspecific, or they exhibit weak charge selectivity (i.e., anion selective or cation selective).

Porins (TC 1.B) are pore-forming proteins that exhibit -barrel structures or variations on the -barrel structural theme. They are localized to the outer membranes of gram-negative bacteria, mitochondria, and chloroplasts. They exhibit a wider range of substrate selectivities than do the -type channel proteins cited above (Table 5). However, like channel protein types, most porins either are nonselective, exhibit some degree of anionic or cationic selectivity, or function in the export of macromolecules across the outer membranes of gram-negative bacteria. Many porins allow passage of any molecule smaller than a certain cutoff size (usually about 500 to 1,000 Da). Of the macromolecular export porin types, more than half export proteins, but several transport complex carbohydrates, and at least one functions in DNA transport.

Examination of the specificities of porins for organic substrates reveals a wide variety of specificities. The maltoporin of E. coli (TC 1.B.3.1.1) and the raffinose porin of E. coli (TC 1.B.15.1.1) are both inducible by their sugar substrates, but while maltoporin is quite specific for maltooligosaccharides, the raffinose porin transports a variety of oligosaccharides. Other porins have been reported to preferentially transport organophosphates (TC 1.B.1.1.2), fatty acids (TC 1.B.9.1.1), nucleosides (TC 1.B.10.1.1), or organic solvents such as toluene (TC 1.B.9.2.1). Still another type apparently exhibits specificity for short-chain amides and urea (TC 1.B.16.1.1). Members of the outer membrane receptor family (TC 1.B.14) import vitamin B₁₂ and a variety of iron-siderophore complexes in a process that is coupled to the PMF via the TonB-dependent energy-coupling system (TC 2.C.1). To what degree these proteins are biochemically selective is not always clear, although they are often encoded within operons that exhibit specific induction properties, and these systems are constituents of transenvelope transport complexes.

Primary carriers are in general highly specific for one or a few related substrates, and like channels, they are almost always selective for inorganic ions or macromolecules. Those specific for organic molecules of small or intermediate sizes belong to either of two superfamilies, the ABC superfamily (TC 3.A.1) or the PTS functional superfamily (TC 4.A.1-6). The PTS is actually a group translocating system, since it phosphorylates its substrates using phosphoenolpyruvate as the phosphoryl donor. For the purpose of tabulating substrate specificities as presented in Table 5, we have grouped this functional superfamily together with the active transporters. The energy-coupling mechanisms used for the transport of ions are diverse, involving pyrophosphate bond hydrolysis, decarboxylation, methyltransfer, oxidoreduction (both hydride shift and electron flow), and light absorption (Fig. 1). By contrast, all macromolecular primary active transporters use ATP or GTP hydrolysis to drive export, although a few macromolecular secondary active exporters use the PMF as the energy source for transport.

Secondary carrier types (TC class 2, subclass 2.A) exhibit a very different spectrum of substrate specificities. None is nonselective or water selective, but many are selective for specific inorganic cations or anions, and a few appear to function in the export of lipids, proteins, or complex carbohydrates, as is characteristic of primary active transporters. Others function in the transport of the many different types of small organic molecules found in biological systems. Thus, every class of molecules included in Table 5 is transported by one or more currently identified secondary carrier(s). For example, members of four transporter families are known to transport sugars and polyols; 13 types transport monocarboxylates, and 12 types transport di- and tricarboxylates. Eighteen types function in the transport of amino acids and their conjugates. It is clear that secondary carriers are primarily responsible for the transport of small organic molecules in virtually all living organisms.

The last column in Table 5 reveals the total numbers of families involved in the transport of the various types of biologically important compounds. About equal numbers of families are concerned with transport of inorganic and organic compounds, with most of the 105 families for inorganic molecules transporting ionic species. Thirty-three families are concerned with carbon source uptake, while 34 are concerned with the uptake of nitrogen-containing compounds (amino acids, bases, and their derivatives). Only 14 families include members that take up compounds in category V (i.e., vitamins, cofactors, signaling molecules, and related compounds), while only 8 families are concerned with transport of hydrophobic substances (category VI). Macromolecules are exported via the transporters of 26 families. While inorganic molecules and macromolecules are transported by all four types of systems, small organic substances are transported almost exclusively by secondary carriers (Table 5).

SUBSTRATE SELECTIVITIES

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Cytoplasmic Membrane Channel Proteins (Excluding Porins)

Table 6 provides a more detailed breakdown of the substrate selectivities of channel-type proteins. The majority of these channel types either are nonselective or merely exhibit a charge preference, preferring inorganic cations over anions or anions over cations. Many of these nonselective channel types are toxic proteins or peptides (CAPs) that are secreted by one cell in order to kill another. Most other channel types exhibit a striking degree of specificity. Seven of these are selective for chloride and other anions, two are specific for Na⁺, and two are selective for K⁺. Five channel types are specific for Ca²⁺. Only MIP and urea transporter family members transport small neutral molecules, as noted above. Five families include members that preferentially transport proteins, but one of these, the holin functional superfamily, consists of 16 families of functionally (but not phylogenetically) similar proteins. The holins (156) and Bcl-2 (1) are bacterial and animal proteins, respectively, that promote cell suicide or apoptosis. Members of the diphtheria toxin and botulinum and tetanus toxin families transport bacterial toxins into target animal cells (69, 86). The proteins of the MscL family may provide protection against osmotic downshift, but they have been shown to be capable of catalyzing protein export as well (2, 8, 12).

Table 7 provides a detailed breakdown of channel-type proteins according to their substrate specificities. The individual families represented in each category are tabulated according to TC number. The name or abbreviation, source, and mode of regulation (when known) are also provided. Among the nonselective channels is the MscL channel of E. coli, which has been reported to exhibit a slight preference for cations over anions and to also transport proteins, while the MscS channel of E. coli has been reported to exhibit a slight anionic preference. Holins function primarily in protein export, while connexins and innexins function to form tight junctions between adjacent animal cells in vertebrates and invertebrates, respectively. Otherwise, all nonselective channel proteins or peptides are designed for export from the cell of synthesis for the purpose of biological warfare. These proteins and peptides are derived from phages, bacteria, and eukaryotes. Archaeal protein and peptide toxins that function by pore formation have not yet been characterized functionally.

Low-specificity cation-selective channels include both acetylcholine- and serotonin-activated ligand-gated ion channel family members, ATP-sensitive ATP-gated cation channel family members, glutamate-gated ion channel family members (all of animal origin), and the MscL family proteins noted above (of bacterial origin). Other cation-selective channels include the members of the nonselective cation channel 1 (NSCC1) and NSCC2 families. Two families, the influenza virus matrix-2 channel and CybB families, selectively transport protons. Still others exhibit selectivity for a particular cation, Na⁺, K⁺, NH₄⁺, or Ca²⁺.

Three of the anion-selective channel types listed appear to transport Cl selectively, although at least three additional families include members that transport other anions as well. Thus, chloride channel (ClC) family proteins transport a variety of inorganic anions, while members of the PLM family transport a wide range of monovalent inorganic and organic anions (Table 7). Three types of ligand-gated channel-forming members of the ligand-gated ion channel family specifically transport chloride, and a high degree of selectivity may be a characteristic of the voltage-regulated organellar ClC (O-ClC) proteins. The organellar ClC family is not related to the ClC family. Proteins of the large and ubiquitous ClC family are found in all three domains of life (bacteria, archaea, and eukarya), but only eukaryotic members have been functionally characterized. The epithelial ClC family includes members that can transport a range of anions.

All characterized integral membrane proteins in the outer (lipopolysaccharide-containing) membranes of gram-negative bacteria are believed to consist largely of -structure rather than -structure, and structural features may provide a targeting signal for the outer membrane (18, 47). Among these proteins are the oligomeric (mostly trimeric) porins, several of which have been structurally characterized (48, 55, 81) (see below). These proteins can transport small molecules nonselectively, or they can be highly selective for a single class of molecules (18, 90, 150). They are found in the outer membranes of mitochondria and plant plastids (11, 40) and may be present in the outer mycolic acid-containing membranes of acid-fast gram-positive bacteria, such as species of mycobacteria and Nocardia (61, 109, 133). Because of their unique subcellular locations and structures, outer membrane -barrel porins are classified separately from the -type channel proteins.

Lipopolysaccharide-containing outer membranes of gram-negative bacteria provide an unusually effective barrier against hydrophobic dyes, detergents, and hydrophobic and amphipathic drugs. However, by virtue of the presence of -barrel-type porins in these structures, the membranes are generally permeable to hydrophilic molecules smaller than 650 Da (91). While some of these porins are essentially nonspecific, others appear to exhibit a high degree of selectivity. Tables 8 and 9 summarize the substrate specificities of various recognized outer membrane porins. All of these proteins are derived from gram-negative bacteria except for members of the mitochondrial and plastid porin family (TC 1.B.8). Table 8 presents the breakdown of substrate selectivities. The conclusions reached are in some cases based on detailed biochemical analyses, but in other cases, physiological data were used to derive the conclusions presented. Thus, not all of the porins represented may prove to be as selective as indicated. A significant percentage of the porins are either nonselective or selective only with respect to the charge of the transported species. Among the anion-selective porins, some function primarily in the transport of phosphate, pyrophosphate, nucleotides, and/or fatty acids. Other small-substrate-selective porins have been reported to exhibit specificity for nucleosides, oligosaccharides, short-chain amides, or toluene. Still other porin-like proteins are designed for the export of drugs and heavy metals, while others function in the import of iron complexes and the vitamin precursor cobalamine. Several porin types apparently function in the export of complex carbohydrates and proteins. In most of these cases, the degree of specificity exhibited by these porins has not been studied extensively.

Table 9 provides a detailed breakdown of porin types according to their physiologically relevant substrate specificities. The individual TC numbers and families are presented, allowing the reader to trace the proteins cited and to identify the primary references so as to be able to examine the experimental evidence concerning their specificities.

Secondary carriers catalyze (i) the transmembrane transport of a single molecular species (uniport), (ii) the cotransport of a solute with a cation (symport), (iii) the countertransport of a solute against a cation (antiport), or (iv) the exchange of one solute for another (solute-solute antiport). Several can catalyze more than one such process (i.e., uniport or symport as well as solute-solute antiport), and single mutations can interconvert uniporters and symporters (143, 151). Some can cotransport several cations while countertransporting other cations. These proteins exhibit a wide variety of topological types and substrate specificities. They are responsible for the transport of most organic solutes across biological membranes, particularly those of eukaryotes that lack nutrient uptake permeases of the ABC superfamily (130).

Table 10 tabulates secondary carriers according to substrate. Secondary carriers are known as those that transport almost any inorganic ion of biological importance. Virtually all inorganic mono-, di-, and trivalent cations as well as a wide variety of biologically important inorganic anions are substrates of these transporters. In addition, all classes of organic molecules are transported by secondary carriers (Table 10). Only a few secondary carriers are believed to function in the export of macromolecules (complex polysaccharides, lipids, and proteins).

Table 11 provides a detailed breakdown of secondary transporters according to substrate, but in contrast to Table 10, Table 11 provides TC number, family, energy-coupling mechanism, and organismal distribution. Monovalent cations are generally transported either in symport with or by antiport against one or more other cations. Thirteen families are primarily concerned with the catalysis of monovalent cation transport. Di- and trivalent cations are probably taken up by uniport or by H⁺ or Na⁺ symport, and efflux is probably mediated by H⁺ antiport. In many of these cases, the energy-coupling mechanism is not w