Microbiol Mol Biol Rev 62(1): 1-34

Major Facilitator Superfamily

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

^{Corresponding author. Mailing address: Department of Biology, University of California at San Diego, La Jolla, CA 92093-0116. Phone: (619) 534-4084. Fax: (619) 534-7108. E-mail:}ude.dscu@reiasm.

^{Present address: School of Biological Sciences, University of Sydney, New South Wales, 2006 Australia.}

Abstract

The major facilitator superfamily (MFS) is one of the two largest families of membrane transporters found on Earth. It is present ubiquitously in bacteria, archaea, and eukarya and includes members that can function by solute uniport, solute/cation symport, solute/cation antiport and/or solute/solute antiport with inwardly and/or outwardly directed polarity. All homologous MFS protein sequences in the public databases as of January 1997 were identified on the basis of sequence similarity and shown to be homologous. Phylogenetic analyses revealed the occurrence of 17 distinct families within the MFS, each of which generally transports a single class of compounds. Compounds transported by MFS permeases include simple sugars, oligosaccharides, inositols, drugs, amino acids, nucleosides, organophosphate esters, Krebs cycle metabolites, and a large variety of organic and inorganic anions and cations. Protein members of some MFS families are found exclusively in bacteria or in eukaryotes, but others are found in bacteria, archaea, and eukaryotes. All permeases of the MFS possess either 12 or 14 putative or established transmembrane α-helical spanners, and evidence is presented substantiating the proposal that an internal tandem gene duplication event gave rise to a primordial MFS protein prior to divergence of the family members. All 17 families are shown to exhibit the common feature of a well-conserved motif present between transmembrane spanners 2 and 3. The analyses reported serve to characterize one of the largest and most diverse families of transport proteins found in living organisms.

Abstract

“If you do not expect to, you will not discover the unexpected.”
Heraclitus

Transport systems allow the uptake of essential nutrients and ions, excretion of end products of metabolism and deleterious substances, and communication between cells and the environment (53). They also provide essential constituents of energy-generating and energy-consuming systems (54). Primary active transporters drive solute accumulation or extrusion by using ATP hydrolysis, photon absorption, electron flow, substrate decarboxylation, or methyl transfer (17). If charged molecules are unidirectionally pumped as a consequence of the consumption of a primary cellular energy source, electrochemical potentials result (54). The consequential chemiosmotic energy generated can then be used to drive the active transport of additional solutes via secondary carriers which merely facilitate the transport of one or more molecular species across the membrane (48, 49).

Recent genome-sequencing data and a wealth of biochemical and molecular genetic investigations have revealed the occurrence of dozens of families of primary and secondary transporters (63). Two such families have been found to occur ubiquitously in all classifications of living organisms. These are the ATP-binding cassette (ABC) superfamily (15, 21, 37, 44) and the major facilitator superfamily (MFS), also called the uniporter-symporter-antiporter family (7, 28, 30, 35, 51). While ABC family permeases are in general multicomponent primary active transporters, capable of transporting both small molecules and macromolecules in response to ATP hydrolysis (59), the MFS transporters are single-polypeptide secondary carriers capable only of transporting small solutes in response to chemiosmotic ion gradients. Although well over 100 families of transporters have now been recognized and classified (73), the ABC superfamily and MFS account for nearly half of the solute transporters encoded within the genomes of microorganisms (63). They are also prevalent in higher organisms. The importance of these two families of transport systems to living organisms can therefore not be overestimated.

The MFS was originally believed to function primarily in the uptake of sugars (36, 46). Subsequent studies revealed that drug efflux systems and Krebs cycle metabolites belong to this family (30, 62). The family was then expanded to include organophosphate:phosphate exchangers and oligosaccharide:H^{symport permeases (}51). Reizer et al. (67) noted that a mammalian phosphate:Na^{symporter is a distant member of this family; Paulsen et al. (}60) subdivided the MFS drug efflux pumps into two phylogenetically distinct families with differing topologies; and Goffeau et al. (26) identified a novel MFS family that consists exclusively of functionally uncharacterized proteins from Saccharomyces cerevisiae revealed by genome sequencing. Recently, Williams and Shaw (86) noted that a family of bacterial aromatic acid permeases belongs to the MFS. These observations led to the probability that the MFS is far more widespread in nature and far more diverse in function than had been thought previously.

Although isolated reports have allowed recognition of an increasing degree of diversity within the MFS, there has been no recent systematic attempt to identify the sequenced proteins that make up the MFS and to classify these proteins into phylogenetic families. We have therefore undertaken this task in the hopes of allowing (i) recognition of the significance of this family to cell physiology; (ii) extrapolation of biochemical, molecular genetic, and biophysical information obtained from the study of a few such systems to all members of the family; (iii) unification of mechanistic models, to the greatest extent possible, so as to be applicable to a maximal number of transporters; (iv) introduction of a rational system of MFS protein classification; and (v) comprehension of the pathways taken in the development of structural and functional diversity resulting from the evolutionary process used.

In this report, we present analyses that allow us to generalize some previous observations regarding the MFS and to note additional characteristics of this immense superfamily. Thus, based exclusively on degrees of sequence similarity, we have constructed phylogenetic trees which allow us to divide all the recognized members of the MFS into 17 families. The members of each family all proved to be more closely related in sequence to each other than they were to any of the other MFS proteins. This fact presumably reflects the evolutionary histories of these proteins (71, 72), and, remarkably, we find that phylogenetic family correlates with function. Thus, each of the families recognizes and transports a distinct class of structurally related compounds. These observations have allowed us to derive a rational classification system for the MFS based on both phylogeny and function. This classification system has proven applicable to virtually all permeases found in nature (73).

In 1990, Rubin et al. (70) presented evidence that strongly argued in favor of an earlier suggestion (see reference 36), that MFS permeases arose by a tandem intragenic duplication event. In this report, we provide additional statistical evidence in favor of this possibility. This event generated the 12-transmembrane-spanner (TMS) protein topology from a primordial 6-TMS unit. Surprisingly, all currently recognized MFS permeases retain the two six-TMS units within a single polypeptide chain, although in 3 of the 17 MFS families, an additional two TMSs are found (60). Moreover, the well-conserved MFS-specific motif between TMS2 and TMS3 and the related but less well conserved motif between TMS8 and TMS9 (36) prove to be a characteristic of virtually all of the more than 300 MFS proteins identified. The functional significance of this repeated motif has been examined by Jessen-Marshall et al. (39) and by Yamaguchi et al. (87–89).

Many additional observations allowed the identification of highly specific characteristics of individual MFS families as well as general characteristics of the MFS as a whole. We hope that the computational analyses reported will provide a guide for molecular biologists, biochemists, and biophysicists interested in structural, functional, and evolutionary aspects of MFS permeases.

ACKNOWLEDGMENTS

We thank Tim Bailey, Charles Elkan, Jayna Ditty, André Goffeau, Caroline Harwood, Gary Kuan, Ellen Neidle, Jonathan Reizer, and Marek Sliwinski for valuable discussions. We also thank Mary Beth Hiller, Lyn Alkan, and Milda Simonaitis for assistance in the preparation of the manuscript.

Work in our laboratory was supported by USPHS grants 2RO1 AI14176 from the National Institute of Allergy and Infectious Diseases and 2RO1 GM55434 from the National Institute of General Medical Science (to M.H.S.). I.T.P. was supported by a C. J. Martin Fellowship from the National Health and Medical Council of Australia.

ACKNOWLEDGMENTS

ADDENDUM IN PROOF

H. Huel, S. Turgut, K. Schmid, and J. W. Lengeler (J. Bacteriol. 179:6014–6019, 1997) have recently reported the sequences of the d-arabinitol:H^{and ribitol:H}^{symport permeases of}Klebsiella pneumoniae (DalT and RbtT, respectively). These two proteins are 86% identical and are 425 and 427 aminoacyl residues long, respectively, both with 12 putative TMSs. We have conducted phylogenetic analyses of these two polyol permeases and have found that they, together with an uncharacterized protein encoded within the Bacillus subtilis genome, comprise a novel MFS family which we have termed the polyol permease (PP) family (family 18) (T.-T. Tseng and M. H. Saier, Jr., unpublished observations). The proteins of the PP family exhibit an approximation to the MFS-specific sequence motif between TMSs 2 and 3 (Table (Table19)19) of GVVAEIIGPRKTM, thus showing poor correspondence to the N-terminal half of this MFS-specific motif but excellent correspondence to the C-terminal half. Binary comparison of DalT with KgtP Eco (Table (Table6)6) gave a comparison score of 10.5 standard deviations for a segment of 107 residues (21% identity, 49% similarity, 0 gaps). This value is sufficient to establish that the three proteins of the PP family are members of the MFS (T.-T. Tseng and M. H. Saier, Jr., unpublished results). The three proteins of the PP family also exhibit recognizable sequence similarity to members of several other MFS permease families. The following two signature sequences proved to be specific to the PP family: Y(A/G)(L/I/V)RGX(A/G)YPLFXYSF(L/I/V)V and GEX₂TLWXALXFX₃GG(L/I/V)₂AL (X is any residue). By hybrid protein construction, Heuel et al. demonstrated that the substrate specificities and kinetic properties for transport of DalT and RbtT are determined by the amino-terminal halves of the proteins. This result contrasts with those reported for the lactose permease of E. coli (LacY; TC 2.1.5.1) in which substrate specificity appears to be determined primarily by residues in the carboxy-terminal half of the protein.

In recent work, M. J. Whipp, H. Camakaris, and A. J. Pittard have cloned and analyzed the shiA gene, which encodes the shikimate transport system of E. coli K-12 (SwissProt accession no. {"type":"entrez-protein","attrs":{"text":"P76350","term_id":"2500934","term_text":"P76350"}}P76350; 438 amino acids) (Gene, in press). This permease proved to be a member of the metabolite:H^{symporter (MHS) family (family 6) of the MFS.}

ADDENDUM IN PROOF

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