The major facilitator superfamily (MFS) revisited

The major facilitator superfamily (MFS) is the largest known superfamily of secondary carriers found in the biosphere. It is ubiquitously distributed throughout virtually all currently recognized organismal phyla. This superfamily currently (2012) consists of 74 families, each of which is usually concerned with the transport of a certain type of substrate. Many of these families, defined phylogenetically, do not include even a single member that is functionally characterized. In this article, we probe the evolutionary origins of these transporters, providing evidence that they arose from a single 2-transmembrane segment (TMS) hairpin structure that triplicated to give a 6-TMS unit that duplicated to a 12-TMS protein, the most frequent topological type of these permeases. We globally examine MFS protein topologies, focusing on exceptional proteins that deviate from the norm. Nine distantly related families appear to have members with 14?TMSs in which the extra two are usually centrally localized between the two 6-TMS repeat units. They probably have arisen by intragenic duplication of an adjacent hairpin. This alternative topology probably arose multiple times during MFS evolution. Convincing evidence for MFS permeases with fewer than 12?TMSs was not forthcoming, leading to the suggestion that all 12?TMSs are required for optimal function. Some homologs appear to have 13, 14, 15 or 16 TMSs, and the probable locations of the extra TMSs were identified. A few MFS permeases are fused to other functional domains or are fully duplicated to give 24-TMS proteins with dual functions. Finally, the MFS families with no known function were subjected to genomic context analyses leading to functional predictions.     

Expansion of the APC superfamily of secondary carriers

The amino acid‐polyamine‐organoCation (APC) superfamily is the second largest superfamily of secondary carriers currently known. In this study, we establish homology between previously recognized APC superfamily members and proteins of seven new families. These families include the PAAP (Putative Amino Acid Permease), LIVCS (Branched Chain Amino Acid:Cation Symporter), NRAMP (Natural Resistance‐Associated Macrophage Protein), CstA (Carbon starvation A protein), KUP (K⁺ Uptake Permease), BenE (Benzoate:H⁺ Virginia Symporter), and AE (Anion Exchanger). The topology of the well‐characterized human Anion Exchanger 1 (AE1) conforms to a UraA‐like topology of 14 TMSs (12 α‐helical TMSs and 2 mixed coil/helical TMSs). All functionally characterized members of the APC superfamily use cation symport for substrate accumulation except for some members of the AE family which frequently use anion:anion exchange. We show how the different topologies fit into the framework of the common LeuT‐like fold, defined earlier (Proteins. 2014 Feb;82(2):336‐46), and determine that some of the new members contain previously undocumented topological variations. All new entries contain the two 5 or 7 TMS APC superfamily repeat units, sometimes with extra TMSs at the ends, the variations being greatest within the CstA family. New, functionally characterized members transport amino acids, peptides, and inorganic anions or cations. Except for anions, these are typical substrates of established APC superfamily members. Active site TMSs are rich in glycyl residues in variable but conserved constellations. This work expands the APC superfamily and our understanding of its topological variations. Proteins 2014; 82:2797–2811. © 2014 Wiley Periodicals, Inc.     

The drug/metabolite transporter superfamily.

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 alpha 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.     

Evolutionary relationship between 5+5 and 7+7 inverted repeat folds within the amino acid‐polyamine‐organocation superfamily

Evidence has been presented that 5+5 TMS and 7+7 TMS inverted repeat fold transporters are members of a single superfamily named the Amino acid‐Polyamine‐organoCation (APC) superfamily. However, the evolutionary relationship between the 5+5 and the 7+7 topological types has not been established. We have identified a common fold, consisting of a spiny membrane helix/sheet, followed by a U‐like structure and a V‐like structure that is recurrent between domain duplicated units of 5+5 and 7+7 inverted repeat folds. This fold is found in the following protein structures: AdiC, ApcT, LeuT, Mhp1, BetP, CaiT, and SglT (all 5+5 TMS repeats), as well as UraA and SulP (7+7 TMS repeats). AdiC, LeuT and Mhp1 have two extra TMSs after the second duplicated domain, SglT has four extra C‐terminal TMSs, and BetP has two extra TMSs before the first duplicated domain. UraA and SulP on the other hand have two extra TMSs at the N‐terminus of each duplicated domain unit. These observations imply that multiple hairpin and domain duplication events occurred during the evolution of the APC superfamily. We suggest that the five TMS architecture was primordial and that families gained two TMSs on either side of this basic structure via dissimilar hairpin duplications either before or after intragenic duplication. Evidence for homology between TMSs 1–2 of AdiC and TMSs 1–2 and 3–4 of UraA suggests that the 7+7 topology arose via an internal duplication of the N‐terminal hairpin loop within the five TMS repeat unit followed by duplication of the 7 TMS domain. Proteins 2014; 82:336–346. © 2013 Wiley Periodicals, Inc.     

MFS超家族转运蛋白结构与分子机制的研究

主要协助转运蛋白超家族（major facilitator superfamily,MFS）是一个主要的次级膜转运蛋白超家族。MFS超家族蛋白转运底物的多样性使得它们在细胞物质交换和能量代谢过程中起着重要作用。从2003年第一个高分辨率的LacY蛋白三维结构的解析到现在,已经有5个细菌MFS超家族的蛋白结构被解析出来,结合大量的生化研究结果,使得对其转运的分子机制有了更为深入的理解。将对MFS超家族蛋白的三维结构和转运机理进行阐述。     

Modelling and mutational evidence identify the substrate binding site and functional elements in APC amino acid transporters

The Amino acid-Polyamine-Organocation (APC) superfamily is the main family of amino acid transporters found in all domains of life and one of the largest families of secondary transporters. Here, using a sensitive homology threading approach and modelling we show that the predicted structure of APC members is extremely similar to the crystal structures of several prokaryotic transporters belonging to evolutionary distinct protein families with different substrate specificities. All of these proteins, despite having no primary amino acid sequence similarity, share a similar structural core, consisting of two V-shaped domains of five transmembrane domains each, intertwined in an antiparallel topology. Based on this model, we reviewed available data on functional mutations in bacterial, fungal and mammalian APCs and obtained novel mutational data, which provide compelling evidence that the amino acid binding pocket is located in the vicinity of the unwound part of two broken helices, in a nearly identical position to the structures of similar transporters. Our analysis is fully supported by the evolutionary conservation and specific amino acid substitutions in the proposed substrate binding domains. Furthermore, it allows predictions concerning residues that might be crucial in determining the specificity profile of APC members. Finally, we show that two cytoplasmic loops constitute important functional elements in APCs. Our work along with different kinetic and specificity profiles of APC members in easily manipulated bacterial and fungal model systems could form a unique framework for combining genetic, in-silico and structural studies, for understanding the function of one of the most important transporter families.     

Topological and phylogenetic analyses of bacterial holin families and superfamilies

Holins are small “hole-forming” transmembrane proteins that mediate bacterial cell lysis during programmed cell death or following phage infection. We have identified fifty two families of established or putative holins and have included representative members of these proteins in the Transporter Classification Database (TCDB; www.tcdb.org). We have identified the organismal sources of members of these families, calculated their average protein sizes, estimated their topologies and determined their relative family sizes. Topological analyses suggest that these proteins can have 1, 2, 3 or 4 transmembrane α-helical segments (TMSs), and members of a single family are frequently, but not always, of a single topology. In one case, proteins of a family proved to have either 2 or 4 TMSs, and the latter arose by intragenic duplication of a primordial 2 TMS protein-encoding gene resembling the former. Using established statistical approaches, some of these families have been shown to be related by common descent. Seven superfamilies, including 21 of the 52 recognized families were identified. Conserved motif and Pfam analyses confirmed most superfamily assignments. These results serve to expand upon the scope of channel-forming bacterial holins.     

Long evolutionary conservation and considerable tissue specificity of several atypical solute carrier transporters

The superfamily of Solute Carriers (SLCs) has around 384 members in the human genome grouped into at least 48 families. While many of these transporters have been well characterized with established important biological functions, there are few recently identified genes that are not studied regarding tissue distribution or evolutionary origin. Here we study 14 of these recently discovered SLC genes (HIAT1, HIATL1, MFSD1, MFSD5, MFSD6, MFSD9, MFSD10, SLC7A14, SLC7A15, SLC10A6, SLC15A5, SLC16A12, SLC30A10 and SLC21A21) with the purpose to give much better picture over the sequence relationship and tissue expression of the diverse SLC gene family. We used a range of bioinformatic methods to classify each of these genes into the different SLC gene families. We found that 9 of the 14 atypical SLCs are distant members of the Major Facilitator Superfamily (MFS) clan while the others belong to the APC clan, the DMT clan, the CPA_AT clan and the IT clan. We found most of the genes to be highly evolutionary conserved, likely to be present in most bilateral species, except for SLC21A21 that we found only present in mammals. Several of these transporter genes have highly specific tissue expression profile while it is notable that most are expressed in the CNS with the exception of SLC21A21 and SLC15A5. This work provides fundamental information on 14 transporters that previously have not received much attention enabling a more comprehensive view over the SLC superfamily.     

The major amino acid transporter superfamily has a similar core structure as Na+-galactose and Na+-leucine transporters

The sodium solute symporters (SSS) and neurotransmitter sodium symporters (NSS) are two families of secondary transporters that are not related in amino acid sequence. Nonetheless, recent crystal structures showed that the Na⁺/galactose (SSS) and Na⁺/leucine (NSS) transporters have similar core structures. The structural relatedness highlights the need for classification methods for membrane protein structures based on other criteria than amino acid similarity. Here, we demonstrate that a method based on hydropathy profile alignments convincingly identifies structural similarity between the NSS and SSS families. Most importantly, the method shows that one of the largest transporter families for which a crystal structure is elusive (the amino acid/polyamine/organocation or APC superfamily), also shares the similar core structure observed for the Na⁺/galactose and Na⁺/leucine transporters. The APC superfamily contains the major amino acid transporter families that are found throughout life. Insight into their structure will significantly facilitate the studies of this important group of transporters.     

Bioinformatic characterization of the 4-Toluene Sulfonate Uptake Permease (TSUP) family of transmembrane proteins

The ubiquitous sequence diverse 4-Toluene Sulfonate Uptake Permease (TSUP) family contains few characterized members and is believed to catalyze the transport of several sulfur-based compounds. Prokaryotic members of the TSUP family outnumber the eukaryotic members substantially, and in prokaryotes, but not eukaryotes, extensive lateral gene transfer occurred during family evolution. Despite unequal representation, homologues from the three taxonomic domains of life share well-conserved motifs. We show that the prototypical eight TMS topology arose from an intragenic duplication of a four transmembrane segment (TMS) unit. Possibly, a two TMS α-helical hairpin structure was the precursor of the 4 TMS repeat unit. Genome context analyses confirmed the proposal of a sulfur-based compound transport role for many TSUP homologues, but functional outliers appear to be prevalent as well. Preliminary results suggest that the TSUP family is a member of a large novel superfamily that includes rhodopsins, integral membrane chaperone proteins, transmembrane electron flow carriers and several transporter families. All of these proteins probably arose via the same pathway: 2→4→8 TMSs followed by loss of a TMS either at the N- or C-terminus, depending on the family, to give the more frequent 7 TMS topology.     

The bile/arsenite/riboflavin transporter (BART) superfamily

Secondary transmembrane transport carriers fall into families and superfamilies allowing prediction of structure and function. Here we describe hundreds of sequenced homologues that belong to six families within a novel superfamily, the bile/arsenite/riboflavin transporter (BART) superfamily, of transport systems and putative signalling proteins. Functional data for members of three of these families are available, and they transport bile salts and other organic anions, the bile acid:Na(+) symporter (BASS) family, inorganic anions such as arsenite and antimonite, the arsenical resistance-3 (Acr3) family, and the riboflavin transporter (RFT) family. The first two of these families, as well as one more family with no functionally characterized members, exhibit a probable 10 transmembrane spanner (TMS) topology that arose from a tandemly duplicated 5 TMS unit. Members of the RFT family have a 5 TMS topology, and are homologous to each of the repeat units in the 10 TMS proteins. The other two families [sensor histidine kinase (SHK) and kinase/phosphatase/synthetase/hydrolase (KPSH)] have a single 5 TMS unit preceded by an N-terminal TMS and followed by a hydrophilic sensor histidine kinase domain (the SHK family) or catalytic domains resembling sensor kinase, phosphatase, cyclic di-GMP synthetase and cyclic di-GMP hydrolase catalytic domains, as well as various noncatalytic domains (the KPSH family). Because functional data are not available for members of the SHK and KPSH families, it is not known if the transporter domains retain transport activity or have evolved exclusive functions in molecular reception and signal transmission. This report presents characteristics of a unique protein superfamily and provides guides for future studies concerning structural, functional and mechanistic properties of its constituent members.     

Topological predictions for integral membrane permeases of the phosphoenolpyruvate:sugar phosphotransferase system

We report bioinformatic analyses of the largest superfamily of integral membrane permeases of the bacterial phosphotransferase system (PTS), the Enzyme IIC constituents of the Glc superfamily. Phylogenetic analyses reveal that this superfamily consists of five equally distant families, the Glucose (Glc), beta-Glucoside (Bgl), Fructose (Fru), Mannitol (Mtl) and Lactose (Lac) families. Average hydropathy, amphipathicity and similarity plots were generated for these five families as well as for the entire superfamily. Charged residue distribution was analyzed, and the most conserved sequence motif, common to all five families, was identified. The results show that the members of all five families exhibit similar average hydropathy plots with regions of average amphipathicity and relative conservation also being similar. Evidence is presented suggesting that the Glucitol (Gut) family of Enzyme IIC constituents is a distant member of the Glc superfamily. Based on our analyses we offer a topological model that resembles, but differs in detail from the two previously proposed models.     

Kinetic and mutational analysis of the Trypanosoma brucei NBT1 nucleobase transporter expressed in Saccharomyces cerevisiae reveals structural similarities between ENT and MFS transporters

Parasitic protozoa are unable to synthesise purines de novo and thus depend on the uptake of nucleosides and nucleobases across their plasma membrane through specific transporters. A number of nucleoside and nucleobase transporters from Trypanosoma brucei brucei and Leishmania major have recently been characterised and shown to belong to the equilibrative nucleoside transporter (ENT) family. A number of studies have demonstrated the functional importance of particular transmembrane segments (TMS) in nucleoside-specific ENT proteins. TbNBT1, one of only three bona fide nucleobase-selective members of the ENT family, has previously been shown to be a high-affinity transporter for purine nucleobases and guanosine. In this study, we use the Saccharomyces cerevisiae expression system to build a biochemical model of how TbNBT1 recognises nucleobases. We next performed random in vitro and site-directed mutagenesis to identify residues critical for TbNBT1 function. The identification of residues likely to contribute to permeant binding, when combined with a structural model of TbNBT1 obtained by homology threading, yield a tentative three-dimensional model of the transporter binding site that is consistent with the binding model emerging from the biochemical data. The model strongly suggests the involvement of TMS5, TMS7 and TMS8 in TbNBT1 function. This situation is very similar to that concerning transporters of the major facilitator superfamily (MFS), one of which was used as a template for the threading. This point raises the possibility that ENT and MFS carriers, despite being considered evolutionarily distinct, might in fact share similar topologies and substrate translocations pathways.     

Phylogeny of multidrug transporters.

We currently recognize five large ubiquitous superfamilies and one small eukaryotic-specific family in which cellular multidrug efflux pumps occur. One, the ABC superfamily, includes members that use ATP hydrolysis to drive drug efflux, but the MFS, RND, MATE and DMT superfamilies include members that are secondary carriers, functioning by drug:H(+)or drug:Na(+)antiport mechanisms. The small MET family seems to be restricted to endosomal membranes of eukaryotes, and only a single such system has been functionally characterized. In this review article, these families of drug transporters are discussed and evaluated from phylogenetic standpoints.     

The ion transporter superfamily

We define a novel superfamily of secondary carriers specific for cationic and anionic compounds, which we have termed the ion transporter (IT) superfamily. Twelve recognized and functionally defined families constitute this superfamily. We provide statistical sequence analyses demonstrating that these families were in fact derived from a common ancestor. Further, we characterize the 12 families in terms of (1) the known substrates transported, (2) the modes of transport and energy coupling mechanisms used, (3) the family sizes (in numbers of sequenced protein members in the current NCBI database), (4) the organismal distributions of the members of each family, (5) the size ranges of the constituent proteins, (6) the predicted topologies of these proteins, and (7) the occurrence of non-homologous auxiliary proteins that may either facilitate or be required for transport. No member of the superfamily is known to function in a capacity other than transport. Proteins in several of the constituent families are shown to have arisen by tandem intragenic duplication events, but topological variation has resulted from a variety of dissimilar genetic fusion, splicing and insertional events. The evolutionary relationships between the members of each family are defined, leading to predictions of functionally relevant orthologous relationships. Some but not all of the families include functionally dissimilar paralogues that arose by early extragenic duplication events.     

中国汉族两个马凡综合征家系FBN1基因的一个新插入突变和一个复发点突变

目的:明确两个中国北方汉族马凡综合征(Marfan syndrome,MFS)家系的临床特点,并对其进行基因诊断。方法:对两个家系进行家系调查和系谱分析,应用聚合酶链式反应-DNA测序方法对原纤维蛋白1基因(Fibrillin-1,FBN1)的所有外显子进行测序。应用Swiss-model、Polyphen-2和SIFT软件对发现的变异位点进行功能预测。结果:两个家系均呈常染色显性遗传特点,在家系1患者中发现一个新的插入突变,即第13外显子1691位碱基处插入碱基A(1691 ins A),导致蛋白在第571位氨基酸处翻译提前终止。此外,在家系2患者中发现一个已知的点突变,即第27外显子第3463位碱基由G变为A(3463 GA),导致第1155位氨基酸由天冬氨酸变为天冬酰胺。这两个变异位点在家系的健康人及50例健康对照中均未出现。功能预测发现这两个变异位点均可能会影响FBN1蛋白的结构或功能。结论:在两个MFS家系中发现一个新插入突变位点(1691 ins A)和一个已知点突变位点(3463 GA),为扩大FBN1基因的突变谱及进一步阐明FBN1基因突变在MFS中的作用提供理论依据。     

The LysE Superfamily of Transport Proteins Involved in Cell Physiology and Pathogenesis

The LysE superfamily consists of transmembrane transport proteins that catalyze export of amino acids, lipids and heavy metal ions. Statistical means were used to show that it includes newly identified families including transporters specific for (1) tellurium, (2) iron/lead, (3) manganese, (4) calcium, (5) nickel/cobalt, (6) amino acids, and (7) peptidoglycolipids as well as (8) one family of transmembrane electron carriers. Internal repeats and conserved motifs were identified, and multiple alignments, phylogenetic trees and average hydropathy, amphipathicity and similarity plots provided evidence that all members of the superfamily derived from a single common 3-TMS precursor peptide via intragenic duplication. Their common origin implies that they share common structural, mechanistic and functional attributes. The transporters of this superfamily play important roles in ionic homeostasis, cell envelope assembly, and protection from excessive cytoplasmic heavy metal/metabolite concentrations. They thus influence the physiology and pathogenesis of numerous microbes, being potential targets of drug action.     

Sequence analyses of cyanobacterial bicarbonate transporters and their homologues

The primary HCO3- uptake system in the cyanobacterium Synecocystis is the Na+-dependent transporter SbtA. SbtA and its homologues were identified and shown to display a common topology of ten transmembrane segments (TMSs). These proved to have arisen by an intragenic duplication event from an ancestral gene encoding a five TMS protein product. A region of SbtA shows sufficient similarity to 10 TMS ABC-type integral membrane transport proteins to suggest a common origin. Phylogenetic analyses of the SbtA family revealed two clusters of cyanobacterial homologues with all non-cyanobacterial family members outside of these two clusters. The tree topology suggests that SbtA family members display multiple transport functions.     

The LysE superfamily: topology of the lysine exporter LysE of Corynebacterium glutamicum, a paradyme for a novel superfamily of transmembrane solute translocators

In Corynebacterium glutamicum the LysE carrier protein exhibits the unique function of exporting L-lysine. We here analyze the membrane topology of LysE, a protein of 236 amino acyl residues, using PhoA- and LacZ-fusions. The amino-terminal end of LysE is located in the cytoplasm whereas the carboxy-terminal end is found in the periplasm. Although 6 hydrophobic domains were identified based on hydropathy analyses, only five transmembrane spanning helices appear to be present. The additional hydrophobic segment may dip into the membrane or be surface localized. We show that LysE is a member of a family of proteins found, for example, in Escherichia coil, Bacillus subtilis, Mycobacterium tuberculosis and Helicobacter pylori. This family, which we have designated the LysE family, is distantly related to two additional protein families which we have designated the YahN and CadD families. These three families, the members of which exhibit similar sizes, hydropathy profiles, and sequence motifs comprise the LysE superfamily. Functionally characterized members of the LysE superfamily export L-lysine, cadmium and possibly quarternary amines. We suggest that LysE superfamily members will prove to catalyze export of a variety of biologically important solutes.