Vamsee S. Reddy

The Transporter Classification Database

The Transporter Classification Database (TCDB; http://www.tcdb.org) serves as a common reference point for transport protein research. The database contains more than 10 000 non-redundant proteins that represent all currently recognized families of transmembrane molecular transport systems. Proteins in TCDB are organized in a five level hierarchical system, where the first two levels are the class and subclass, the second two are the family and subfamily, and the last one is the transport system. Superfamilies that contain multiple families are included as hyperlinks to the five tier TC hierarchy. TCDB includes proteins from all types of living organisms and is the only transporter classification system that is both universal and recognized by the International Union of Biochemistry and Molecular Biology. It has been expanded by manual curation, contains extensive text descriptions providing structural, functional, mechanistic and evolutionary information, is supported by unique software and is interconnected to many other relevant databases. TCDB is of increasing usefulness to the international scientific community and can serve as a model for the expansion of database technologies. This manuscript describes an update of the database descriptions previously featured in NAR database issues.

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.

The Amino Acid-Polyamine-Organocation Superfamily

The amino acid-polyamine-organocation (APC) superfamily has been shown to include five recognized families, four of which are specific for amino acids and their derivatives. Recent high-resolution X-ray crystallographic data have shown that four additional transporter families (BCCT, TC No. 2.A.15; SSS, 2.A.21; NSS, 2.A.22; and NCS1, 2.A.39), transporting a wide range of solutes, exhibit sufficiently similar folds to suggest a common evolutionary origin. We have used established statistical methods, based on sequence similarity, to show that these families are, in fact, members of the APC superfamily. We also identify two additional families (NCS2, 2.A.40; SulP, 2.A.53) as being members of this superfamily. Repeat sequences, each having five transmembrane α-helical segments and arising via ancient intragenic duplications, are demonstrated for all of these families, further strengthening the conclusion of homology. The APC superfamily appears to be the second largest superfamily of secondary carriers, the largest being the major facilitator superfamily (MFS). Although the topology of the members of the APC superfamily differs from that of the MFS, both families appear to have arisen from a common ancestral 2 TMS hairpin structure that underwent intragenic triplication followed by loss of a TMS in the APC family, to give the repeat units that are characteristic of these two superfamilies.

BioV Suite--a collection of programs for the study of transport protein evolution.

The Bio‐V Suite is a collection of Python scripts designed specifically for bioinformatic research regarding transport protein evolution. The Bio‐V Suite contains nine powerful programs for Unix‐based environments, each of which can be run as a standalone tool or be accessed in a programmatic manner. These programs and their functions are as follows. The transmembrane alpha‐helical statistical prediction tool (TMStats) generates topological statistics for transport proteins. The Global Sequence Alignment Tool (GSAT) performs shuffle‐based binary alignments and is fully scalable. It can cross‐compare two FASTA files or individual sequences. Protocol1 performs remote PSI‐Blast searches and filters redundant/similar sequences and annotates them. Protocol2 finds homologues between FASTA lists and generates graphical reports. The Targeted Smith–Waterman Search (TSSearch) uses a rapid search algorithm to find distant homologues in FASTA files in a heuristic manner. SSearch is the exhaustive version of TSSearch. Genome‐Blast (GBlast) will identify potential transport proteins in any genome/proteome file or find similar transport protein homologues between two different genomes/proteomes before generating a graphical report. AncientRep (AR) will find putative transmembrane repeat units using a list of homologues. DefineFamily (DF) will generate a FASTA list to represent an entire Transporter Classification family. These nine programs are tabulated with descriptions of their capabilities in Table 1 .

Phylogenetic Characterization of Transport Protein Superfamilies: Superiority of SuperfamilyTree Programs over Those Based on Multiple Alignments

Transport proteins function in the translocation of ions, solutes and macromolecules across cellular and organellar membranes. These integral membrane proteins fall into >600 families as tabulated in the Transporter Classification Database (www.tcdb.org). Recent studies, some of which are reported here, define distant phylogenetic relationships between families with the creation of superfamilies. Several of these are analyzed using a novel set of programs designed to allow reliable prediction of phylogenetic trees when sequence divergence is too great to allow the use of multiple alignments. These new programs, called SuperfamilyTree1 and 2 (SFT1 and 2), allow display of protein and family relationships, respectively, based on thousands of comparative BLAST scores rather than multiple alignments. Superfamilies analyzed include: (1) Aerolysins, (2) RTX Toxins, (3) Defensins, (4) Ion Transporters, (5) Bile/Arsenite/Riboflavin Transporters, (6) Cation:Proton Antiporters, and (7) the Glucose/Fructose/Lactose superfamily within the prokaryotic phosphoenol pyruvate-dependent Phosphotransferase System. In addition to defining the phylogenetic relationships of the proteins and families within these seven superfamilies, evidence is provided showing that the SFT programs outperform programs that are based on multiple alignments whenever sequence divergence of superfamily members is extensive. The SFT programs should be applicable to virtually any superfamily of proteins or nucleic acids.

The Transporter-Opsin-G protein-coupled receptor (TOG) Superfamily

Visual rhodopsins are recognized members of the large and diverse family of G protein‐coupled receptors (GPCRs), but their evolutionary origin and relationships to other proteins are not known. In a previous paper [Shlykov MA, Zheng WH, Chen JS & Saier MH Jr (2012) Biochim Biophys Acta 1818, 703–717], we characterized the 4‐toluene sulfonate uptake permease (TSUP) family of transmembrane proteins, and showed that these 7‐transmembrane segment (TMS) or 8‐TMS proteins arose by intragenic duplication of a gene encoding a 4‐TMS protein, sometimes followed by loss of a terminal TMS. In this study, we show that the TSUP, GPCR and microbial rhodopsin families are related to each other and to six other currently recognized transport protein families. We designate this superfamily the transporter/opsin/G protein‐coupled receptor (TOG) superfamily. Despite their 8‐TMS origins, the members of most constituent families exhibit 7‐TMS topologies that are well conserved, and these arose by loss of either the N‐terminal TMS (more frequent) or the C‐terminal TMS (less frequent), depending on the family. Phylogenetic analyses revealed familial relationships within the superfamily and protein relationships within each of the nine families. The results of the statistical analyses leading to the conclusion of homology were confirmed using hidden Markov models, Pfam and 3D superimpositions. Proteins functioning by dissimilar mechanisms (channels, primary active transporters, secondary active transporters, group translocators and receptors) are interspersed on a phylogenetic tree of the TOG superfamily, suggesting that changes in the transport and energy‐coupling mechanisms occurred multiple times during evolution of this superfamily.

Evolutionary relationships of ATP-Binding Cassette (ABC) uptake porters

BackgroundThe ATP-Binding Cassette (ABC) functional superfamily includes integral transmembrane exporters that have evolved three times independently, forming three families termed ABC1, ABC2 and ABC3, upon which monophyletic ATPases have been superimposed for energy-coupling purposes [e.g., J Membr Biol 231(1):1-10, 2009]. The goal of the work reported in this communication was to understand how the integral membrane constituents of ABC uptake transporters with different numbers of predicted or established transmembrane segments (TMSs) evolved. In a few cases, high resolution 3-dimensional structures were available, and in these cases, their structures plus primary sequence analyses allowed us to predict evolutionary pathways of origin.ResultsAll of the 35 currently recognized families of ABC uptake proteins except for one (family 21) were shown to be homologous using quantitative statistical methods. These methods involved using established programs that compare native protein sequences with each other, after having compared each sequence with thousands of its own shuffled sequences, to gain evidence for homology. Topological analyses suggested that these porters contain numbers of TMSs ranging from four or five to twenty. Intragenic duplication events occurred multiple times during the evolution of these porters. They originated from a simple primordial protein containing 3 TMSs which duplicated to 6 TMSs, and then produced porters of the various topologies via insertions, deletions and further duplications. Except for family 21 which proved to be related to ABC1 exporters, they are all related to members of the previously identified ABC2 exporter family. Duplications that occurred in addition to the primordial 3 → 6 duplication included 5 → 10, 6 → 12 and 10 → 20 TMSs. In one case, protein topologies were uncertain as different programs gave discrepant predictions. It could not be concluded with certainty whether a 4 TMS ancestral protein or a 5 TMS ancestral protein duplicated to give an 8 or a 10 TMS protein. Evidence is presented suggesting but not proving that the 2TMS repeat unit in ABC1 porters derived from the two central TMSs of ABC2 porters. These results provide structural information and plausible evolutionary pathways for the appearance of most integral membrane constituents of ABC uptake transport systems.ConclusionsAlmost all integral membrane uptake porters of the ABC superfamily belong to the ABC2 family, previously established for exporters. Most of these proteins can have 5, 6, 10, 12 or 20 TMSs per polypeptide chain. Evolutionary pathways for their appearance are proposed.

Comparative genomics of transport proteins in developmental bacteria: Myxococcus xanthus and Streptomyces coelicolor

BackgroundTwo of the largest fully sequenced prokaryotic genomes are those of the actinobacterium, Streptomyces coelicolor (Sco), and the δ-proteobacterium, Myxococcus xanthus (Mxa), both differentiating, sporulating, antibiotic producing, soil microbes. Although the genomes of Sco and Mxa are the same size (~9 Mbp), Sco has 10% more genes that are on average 10% smaller than those in Mxa.ResultsSurprisingly, Sco has 93% more identifiable transport proteins than Mxa. This is because Sco has amplified several specific types of its transport protein genes, while Mxa has done so to a much lesser extent. Amplification is substrate- and family-specific. For example, Sco but not Mxa has amplified its voltage-gated ion channels but not its aquaporins and mechano-sensitive channels. Sco but not Mxa has also amplified drug efflux pumps of the DHA2 Family of the Major Facilitator Superfamily (MFS) (49 versus 6), amino acid transporters of the APC Family (17 versus 2), ABC-type sugar transport proteins (85 versus 6), and organic anion transporters of several families. Sco has not amplified most other types of transporters. Mxa has selectively amplified one family of macrolid exporters relative to Sco (16 versus 1), consistent with the observation that Mxa makes more macrolids than does Sco.ConclusionsExcept for electron transport carriers, there is a poor correlation between the types of transporters found in these two organisms, suggesting that their solutions to differentiative and metabolic needs evolved independently. A number of unexpected and surprising observations are presented, and predictions are made regarding the physiological functions of recognizable transporters as well as the existence of yet to be discovered transport systems in these two important model organisms and their relatives. The results provide insight into the evolutionary processes by which two dissimilar prokaryotes evolved complexity, particularly through selective chromosomal gene amplification.