Eric I. Sun

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.

Membrane porters of ATP-binding cassette transport systems are polyphyletic.

The ATP-binding cassette (ABC) superfamily consists of both importers and exporters. These transporters have, by tradition, been classified according to the ATP hydrolyzing constituents, which are monophyletic. The evolutionary origins of the transmembrane porter proteins/domains are not known. Using five distinct computer programs, we here provide convincing statistical data suggesting that the transmembrane domains of ABC exporters are polyphyletic, having arisen at least three times independently. ABC1 porters arose by intragenic triplication of a primordial two-transmembrane segment (TMS)-encoding genetic element, yielding six TMS proteins. ABC2 porters arose by intragenic duplication of a dissimilar primordial three-TMS-encoding genetic element, yielding a distinctive protein family, nonhomologous to the ABC1 proteins. ABC3 porters arose by duplication of a primordial four-TMS-encoding genetic element, yielding either eight- or 10-TMS proteins. We assign each of 48 of the 50 currently recognized families of ABC exporters to one of the three evolutionarily distinct ABC types. Currently available high-resolution structural data for ABC porters are fully consistent with our findings. These results provide guides for future structural and mechanistic studies of these important transport systems.The ATP-binding cassette (ABC) superfamily consists of both importers and exporters. These transporters have, by tradition, been classified according to the ATP hydrolyzing constituents, which are monophyletic. The evolutionary origins of the transmembrane porter proteins/domains are not known. Using five distinct computer programs, we here provide convincing statistical data suggesting that the transmembrane domains of ABC exporters are polyphyletic, having arisen at least three times independently. ABC1 porters arose by intragenic triplication of a primordial two-transmembrane segment (TMS)-encoding genetic element, yielding six TMS proteins. ABC2 porters arose by intragenic duplication of a dissimilar primordial three-TMS-encoding genetic element, yielding a distinctive protein family, nonhomologous to the ABC1 proteins. ABC3 porters arose by duplication of a primordial four-TMS-encoding genetic element, yielding either eight- or 10-TMS proteins. We assign each of 48 of the 50 currently recognized families of ABC exporters to one of the three evolutionarily distinct ABC types. Currently available high-resolution structural data for ABC porters are fully consistent with our findings. These results provide guides for future structural and mechanistic studies of these important transport systems.

Multidrug resistance: phylogenetic characterization of superfamilies of secondary carriers that include drug exporters.

We here describe the application of novel programs that allow definition of phylogenetic relationships in transport protein superfamilies. These programs are used to provide information about the four major superfamilies of secondary carriers that include members that export hydrophobic and amphipathic compounds including drugs. These novel programs must be used when sequence divergence among superfamily members is too great to allow construction of reliable multiple alignments. We test the validity and demonstrate the reliability of these trees by conducting comparative analyses. We examine all of the largest superfamilies of secondary drug efflux pumps found in nature, the MOP, DMT, RND, and MFS superfamilies. Depending on the superfamily, phylogenetic clustering of the families and individual members of these families can occur according to organismal source, substrate type, polarity of transport, and/or mode of transport. In this chapter we define the phylogenetic relationships of sequence divergent drug exporters. The programs developed should be applicable to all classes of proteins and nucleic acids.

Comparative genomics of metabolic capacities of regulons controlled by cis-regulatory RNA motifs in bacteria

BackgroundIn silico comparative genomics approaches have been efficiently used for functional prediction and reconstruction of metabolic and regulatory networks. Riboswitches are metabolite-sensing structures often found in bacterial mRNA leaders controlling gene expression on transcriptional or translational levels.An increasing number of riboswitches and other cis-regulatory RNAs have been recently classified into numerous RNA families in the Rfam database. High conservation of these RNA motifs provides a unique advantage for their genomic identification and comparative analysis.ResultsA comparative genomics approach implemented in the RegPredict tool was used for reconstruction and functional annotation of regulons controlled by RNAs from 43 Rfam families in diverse taxonomic groups of Bacteria. The inferred regulons include ~5200 cis-regulatory RNAs and more than 12000 target genes in 255 microbial genomes. All predicted RNA-regulated genes were classified into specific and overall functional categories. Analysis of taxonomic distribution of these categories allowed us to establish major functional preferences for each analyzed cis-regulatory RNA motif family. Overall, most RNA motif regulons showed predictable functional content in accordance with their experimentally established effector ligands. Our results suggest that some RNA motifs (including thiamin pyrophosphate and cobalamin riboswitches that control the cofactor metabolism) are widespread and likely originated from the last common ancestor of all bacteria. However, many more analyzed RNA motifs are restricted to a narrow taxonomic group of bacteria and likely represent more recent evolutionary innovations.ConclusionsThe reconstructed regulatory networks for major known RNA motifs substantially expand the existing knowledge of transcriptional regulation in bacteria. The inferred regulons can be used for genetic experiments, functional annotations of genes, metabolic reconstruction and evolutionary analysis. The obtained genome-wide collection of reference RNA motif regulons is available in the RegPrecise database (http://regprecise.lbl.gov/).

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.

Genetic Implication of a Novel Thiamine Transporter in Human Hypertension

OBJECTIVES This study coupled 2 strategies-trait extremes and genome-wide pooling-to discover a novel blood pressure (BP) locus that encodes a previously uncharacterized thiamine transporter. BACKGROUND Hypertension is a heritable trait that remains the most potent and widespread cardiovascular risk factor, although details of its genetic determination are poorly understood. METHODS Representative genomic deoxyribonucleic acid (DNA) pools were created from male and female subjects in the highest- and lowest-fifth percentiles of BP in a primary care population of >50,000 patients. The peak associated single-nucleotide polymorphisms were typed in individual DNA samples, as well as in twins/siblings phenotyped for cardiovascular and autonomic traits. Biochemical properties of the associated transporter were evaluated in cellular assays. RESULTS After chip hybridization and calculation of relative allele scores, the peak associations were typed in individual samples, revealing an association between hypertension, systolic BP, and diastolic BP and the previously uncharacterized solute carrier SLC35F3. The BP genetic association at SLC35F3 was validated by meta-analysis in an independent sample from the original source population, as well as the International Consortium for Blood Pressure Genome-Wide Association Studies (across North America and western Europe). Sequence homology to a putative yeast thiamine (vitamin B1) transporter prompted us to express human SLC35F3 in Escherichia coli, which catalyzed [(3)H]-thiamine uptake. SLC35F3 risk-allele homozygotes (T/T) displayed decreased erythrocyte thiamine content on microbiological assay. In twin pairs, the SLC35F3 risk allele predicted heritable cardiovascular traits previously associated with thiamine deficiency, including elevated cardiac stroke volume with decreased vascular resistance, and elevated pressor responses to environmental (cold) stress. Allelic expression imbalance confirmed that cis variation at the human SLC35F3 locus influenced expression of that gene, and the allelic expression imbalance peak coincided with the hypertension peak. CONCLUSIONS Novel strategies were coupled to position a new hypertension-susceptibility locus, uncovering a previously unsuspected thiamine transporter whose genetic variants predicted several disturbances in cardiac and autonomic function. The results have implications for the pathogenesis and treatment of systemic hypertension.

Molecular archeological studies of transmembrane transport systems

We here review studies concerned with the evolutionary pathways taken for the appearance of complex transport systems. The transmembrane protein constituents of these systems generally arose by (1) intragenic duplications, (2) gene fusions, and (3) the superimposition of enzymes onto carriers. In a few instances, we have documented examples of “reverse” or “retrograde” evolution where complex carriers have apparently lost parts of their polypeptide chains to give rise to simpler channels. Some functional superfamilies of transporters that are energized by adenosine triphosphate (ATP) or phosphoenolpyruvate (PEP) include several independently evolving permease families. The ubiquitous ATP-binding cassette (ABC) superfamily couples transport to ATP hydrolysis where the ATPases are superimposed on at least three distinct, independently evolving families of permeases. The prokaryotic sugar transporting phosphotransferase system (PTS) uses homologous PEP-dependent general energy-coupling phosphoryl transfer enzymes superimposed on at least three independently arising families of permeases to give rise to complex group translocators that modify their sugar substrates during transport, releasing cytoplasmic sugar phosphates. We suggest that simple carriers evolved independently of the energizing enzymes, and that chemical energization of transport resulted from the physical and functional coupling of the enzymes to the carriers.