Genome archeology leading to the characterization and classification of transport proteins.

In the study of transmembrane transport, molecular phylogeny provides a reliable guide to protein structure, catalytic and noncatalytic transport mechanisms, mode of energy coupling and substrate specificity. It also allows prediction of the evolutionary history of a transporter family, leading to estimations of its age, source, and route of appearance. Phylogenetic analyses, therefore, provide a rational basis for the characterization and classification of transporters. A universal classification system has been described, based on both function and phylogeny, which has been designed to be applicable to all currently recognized and yet-to-be discovered transport proteins found in living organisms on Earth.     

Classification of 29 families of secondary transport proteins into a single structural class using hydropathy profile analysis

A classification scheme for membrane proteins is proposed that clusters families of proteins into structural classes based on hydropathy profile analysis. The averaged hydropathy profiles of protein families are taken as fingerprints of the 3D structure of the proteins and, therefore, are able to detect more distant evolutionary relationships than amino acid sequences. A procedure was developed in which hydropathy profile analysis is used initially as a filter in a BLAST search of the NCBI protein database. The strength of the procedure is demonstrated by the classification of 29 families of secondary transporters into a single structural class, termed ST[3]. An exhaustive search of the database revealed that the 29 families contain 568 unique sequences. The proteins are predominantly from prokaryotic origin and most of the characterized transporters in ST[3] transport organic and inorganic anions and a smaller number are Na(+)/H(+) antiporters. All modes of energy coupling (symport, antiport, uniport) are found in structural class ST[3]. The relevance of the classification for structure/function prediction of uncharacterised transporters in the class is discussed.     

Comparative analysis of the ADAM and ADAMTS families

The "A Disintegrin And Metalloproteinase" (ADAM) protein family and the "A Disintegrin-like And Metalloproteinase with ThromboSpondin motifs" (ADAMTS) protein family are two related families of human proteins. The similarities and differences between these two families have been investigated using phylogenetic trees and homology modeling. The phylogenetic analysis indicates that the two families are well differentiated, even when only the common metalloprotease domain is taken into account. Within the ADAM family, several proteins are lacking the binding motif for the catalytic zinc in the active site and thus presumably lack any catalytic activity. These proteins tend to cluster within the ADAM phylogenetic tree and are expressed in specific tissues, suggesting a functional differentiation. The present analysis allows us to propose the following: (i) ADAMTS proteins have a conserved role in the human organism as proteases, with some differentiation in terms of substrate specificity; (ii) ADAM proteins can act as proteases and/or mediators of intermolecular interactions; (iii) proteolytically active ADAMs tend to be more ubiquitously expressed than the inactive ones.     

The Transporter Classification (TC) System, 2002

The Transporter Classification (TC) system is a functional/phylogenetic system designed for the classification of all transmembrane transport proteins found in living organisms on Earth. It parallels but differs from the strictly functional EC system developed decades ago by the Enzyme Commission of the International Union of Biochemistry and Molecular Biology (IUBMB) for the classification of enzymes. Recently, the TC system has been adopted by the IUBMB as the internationally acclaimed system for the classification of transporters. Here we present the characteristics of the nearly 400 families of transport systems included in the TC system and provide statistical analyses of these families and their constituent proteins. Specifically, we analyze the transporter types for size and topological differences and analyze the families for the numbers and organismal sources of their constituent members. We show that channels and carriers exhibit distinctive structural and topological features. Bacterial-specific families outnumber eukaryotic-specific families about 2 to 1, while ubiquitous families, found in all three domains of life, are about half as numerous as eukaryotic-specific families. The results argue against appreciable horizontal transfer of genes encoding transporters between the three domains of life over the last 2 billion years.     

Characterization of the citrate/acetate antiporter CitW of Klebsiella pneumoniae

The genome of Klebsiella pneumoniae contains at least three different genes encoding citrate transporters. Recently, a third and hitherto unknown gene encoding a citrate transport system ( citW) was identified. Escherichia coli transformed with a plasmid expressing citW was able to grow on citrate as sole carbon and energy source, identifying CitW as a citrate carrier. In this report, we provide evidence that further specifies CitW as a Na(+)-independent citrate/citrate and citrate/acetate exchanger. Kinetic analysis of citrate uptake at different pH values identified Hcitrate(2-) as the transported citrate species, with a K(m) of 25 microM. Since citW is expressed under anoxic conditions and acetate is the main end-product of citrate fermentation in K. pneumoniae, citrate/acetate exchange might be its in vivo function. Sequence similarity searches identified CitW (454 amino acids, 48.15 kDa) as a member of the 2-hydroxycarboxylate transporter family (TC 2.A.24). The substrate specificity seems to partially contradict this phylogenetic classification, but appears logical with respect to the putative functional role of CitW in the citrate fermentation pathway of K. pneumoniae.     

Bicarbonate transport proteins.

Bicarbonate is not freely permeable to membranes. Yet, bicarbonate must be moved across membranes, as part of CO2 metabolism and to regulate cell pH. Mammalian cells ubiquitously express bicarbonate transport proteins to facilitate the transmembrane bicarbonate flux. These bicarbonate transporters, which function by different transport mechanisms, together catalyse transmembrane bicarbonate movement. Recent advances have allowed the identification of several new bicarbonate transporter genes. Bicarbonate transporters cluster into two separate families: (i) the anion exachanger (AE) family of Cl-/HCO3- exchangers is related in sequence to the NBC family of Na+/HCO3- cotransporters and the Na(+)-dependent Cl/HCO3- exchangers and (ii) some members of the SLC26a family of sulfate transporters will also transport bicarbonate but are not related in sequence to the AE/NBC family of transporters. This review summarizes our understanding of the mammalian bicarbonate transporter superfamily.     

The transporter classification (TC) system, 2002

The Transporter Classification (TC) system is a functional/phylogenetic system designed for the classification of all transmembrane transport proteins found in living organisms on Earth. It parallels but differs from the strictly functional EC system developed decades ago by the Enzyme Commission of the International Union of Biochemistry and Molecular Biology (IUBMB) for the classification of enzymes. Recently, the TC system has been adopted by the IUBMB as the internationally acclaimed system for the classification of transporters. Here we present the characteristics of the nearly 400 families of transport systems included in the TC system and provide statistical analyses of these families and their constituent proteins. Specifically, we analyze the transporter types for size and topological differences and analyze the families for the numbers and organismal sources of their constituent members. We show that channels and carriers exhibit distinctive structural and topological features. Bacterial-specific families outnumber eukaryotic-specific families about 2 to 1, while ubiquitous families, found in all three domains of life, are about half as numerous as eukaryotic-specific families. The results argue against appreciable horizontal transfer of genes encoding transporters between the three domains of life over the last 2 billion years.     

The ABC gene family in arthropods: Comparative genomics and role in insecticide transport and resistance

About a 100 years ago, the Drosophila white mutant marked the birth of Drosophila genetics. The white gene turned out to encode the first well studied ABC transporter in arthropods. The ABC gene family is now recognized as one of the largest transporter families in all kingdoms of life. The majority of ABC proteins function as primary-active transporters that bind and hydrolyze ATP while transporting a large diversity of substrates across lipid membranes. Although extremely well studied in vertebrates for their role in drug resistance, less is known about the role of this family in the transport of endogenous and exogenous substances in arthropods. The ABC families of five insect species, a crustacean and a chelicerate have been annotated in some detail. We conducted a thorough phylogenetic analysis of the seven arthropod and human ABC protein subfamilies, to infer orthologous relationships that might suggest conserved function. Most orthologous relationships were found in the ABCB half transporter, ABCD, ABCE and ABCF subfamilies, but specific expansions within species and lineages are frequently observed and discussed. We next surveyed the role of ABC transporters in the transport of xenobiotics/plant allelochemicals and their involvement in insecticide resistance. The involvement of ABC transporters in xenobiotic resistance in arthropods is historically not well documented, but an increasing number of studies using unbiased differential gene expression analysis now points to their importance. We give an overview of methods that can be used to link ABC transporters to resistance. ABC proteins have also recently been implicated in the mode of action and resistance to Bt toxins in Lepidoptera. Given the enormous interest in Bt toxicology in transgenic crops, such findings will provide an impetus to further reveal the role of ABC transporters in arthropods.     

Origin of tetracycline efflux proteins: conclusions from nucleotide sequence analysis

The sequences of six tetracycline efflux proteins and three transport proteins which have some resemblance to them were compared. The tetracycline efflux proteins fall into three families: (i) those encoded by pBR322, RP1, and Tn10 (Escherichia coli); (ii) pT181 (Staphylococcus aureus) and pTHT15 (Bacillus subtilis); and (iii) tet347 (Streptomyces rimosus). There is global sequence homology within each of the first two families, but there is none between the families. The pT181/pTHT15 family shares close homology with the N-terminal half of the methylenomycin A efflux protein (Streptomyces coelicor), while tet347 resembles the C-terminal half. Portions of the N-terminal half of the Tn10-encoded protein show significant resemblance to portions in the N-terminal half of the pT181/pTHT15 family, but this sometimes occurs among transport proteins which do not have a common substrate. Tetracycline efflux proteins, therefore, appear to have arisen on at least two, or possibly three, separate occasions, probably from other transport proteins.     

A molecular phylogeny of the Cephalaspidea sensu lato (Gastropoda: Euthyneura): Architectibranchia redefined and Runcinacea reinstated

The monophyly and phylogenetic relationships of the Cephalaspidea sensu lato ( sensu   Burn and Thompson 1998 ) have been investigated by means of Bayesian, parsimony and distance analyses of nuclear (18S rRNA and 28S rRNA) and mitochondrial cytochrome oxidase I (COI) genes.
Results revealed the presence of three monophyletic groups among the Cephalaspidea s. l. (i) Architectibranchia ( sensu   Haszprunar 1985 , in part: including Acteonidae and Aplustridae, but excluding Diaphanidae), (ii) Cephalaspidea including Diaphanidae but not Runcinidae (both previously of uncertain systematic affinity), and (iii) Runcinacea.
The monophyly of the architectibranch families Acteonidae (represented by Acteon and Pupa ) and Aplustridae ( Hydatina and Micromelo ); of the runcinacean family Runcinidae ( Runcina ); and of the cephalaspidean families Aglajidae ( Chelidonura , Aglaja , Odontoglaja , Navanax and Philinopsis ), Bullidae ( Bulla ), Gastropteridae ( Siphopteron and Sagaminopteron ), Haminoeidae ( Atys , Haminoea , Phanerophthalmus and Smaragdinella, but not Ventomnestia ), and Retusidae ( Retusa and Pyrunculus , but not Volvulella ) is suggested. The families Scaphandridae ( Scaphander ) and Rhizoridae ( Volvulella ) are reinstated as valid. A new phylogenetic classification of the Cephalaspidea is proposed.     

Microbial genome analyses: comparative transport capabilities in eighteen prokaryotes

Here, we present a comprehensive analysis of solute transport systems encoded within the completely sequenced genomes of 18 prokaryotic organisms. These organisms include four Gram-positive bacteria, seven Gram-negative bacteria, two spirochetes, one cyanobacterium and four archaea. Membrane proteins are analyzed in terms of putative membrane topology, and the recognized transport systems are classified into 76 families, including four families of channel proteins, four families of primary carriers, 54 families of secondary carriers, six families of group translocators, and eight unclassified families. These families are analyzed in terms of the paralogous and orthologous relationships of their protein members, the substrate specificities of their constituent transporters and their distributions in each of the 18 organisms studied. The families vary from large superfamilies with hundreds of represented members, to small families with only one or a few members. The mode of transport generally correlates with the primary mechanism of energy generation, and the numbers of secondary transporters relative to primary transporters are roughly proportional to the total numbers of primary H(+) and Na(+) pumps in the cell. The phosphotransferase system is less prevalent in the analyzed bacteria than previously thought (only six of 14 bacteria transport sugars via this system) and is completely lacking in archaea and eukaryotes. Escherichia coli is shown to be exceptionally broad in its transport capabilities and therefore, at a membrane transport level, does not appear representative of the bacteria thus far sequenced. Archaea and spirochetes exhibit fewer proteins with multiple transmembrane segments and fewer net transporters than most bacteria. These results provide insight into the relevance of transport to the overall physiology of prokaryotes.     

Sugar transport in Sulfolobus solfataricus is mediated by two families of binding protein-dependent ABC transporters

The extreme thermoacidophilic archaeon Sulfolobus solfataricus grows optimally at 80 degrees C and pH 3 and uses a variety of sugars as sole carbon and energy source. Glucose transport in this organism is mediated by a high-affinity binding protein-dependent ATP-binding cassette (ABC) transporter. Sugar-binding studies revealed the presence of four additional membrane-bound binding proteins for arabinose, cellobiose, maltose and trehalose. These glycosylated binding proteins are subunits of ABC transporters that fall into two distinct groups: (i) monosaccharide transporters that are homologous to the sugar transport family containing a single ATPase and a periplasmic-binding protein that is processed at an unusual site at its amino-terminus; (ii) di- and oligosaccharide transporters, which are homologous to the family of oligo/dipeptide transporters that contain two different ATPases, and a binding protein that is synthesized with a typical bacterial signal sequence. The latter family has not been implicated in sugar transport before. These data indicate that binding protein-dependent transport is the predominant mechanism of transport for sugars in S. solfataricus.     

Two novel families of bacterial membrane proteins concerned with nodulation, cell division and transport

Homology has been established for members of two families of functionally related bacterial membrane proteins. The first family (the resistance/nodulation/cell division (RND) family) Includes (i) two metal-resistance efflux pumps in Alcaligenes eutrophus (CzcA and CnrA), (ii) three proteins which function together in nodulation of alfalfa roots by Rhizobium meliloti (NoIGHI), and (iii) a cell division protein in Escherichia coli (EnvD). The second family (the putative membrane fusion protein (MFP) family) includes a nodulation protein (NoIF), a cell division protein (EnvC), and a multidrug resistance transport protein (EmrA). We propose that an MFP functions co-operatively with an RND protein to transport large or hydrophobic molecules across the two membranes of the Gram-negative bacterial cell envelope.     

Molecular basis of essential amino acid transport from studies of insect nutrient amino acid transporters of the SLC6 family (NAT-SLC6)

Two protein families that represent major components of essential amino acid transport in insects have been identified. They are annotated as the SLC6 and SLC7 families of transporters according to phylogenetic proximity to characterized amino acid transporters (HUGO nomenclature). Members of these families have been identified as important apical and basolateral parts of transepithelial essential amino acid absorption in the metazoan alimentary canal. Synergistically, they play critical physiological roles as essential substrate providers to diverse metabolic processes, including generic protein synthesis. This review briefly clarifies the requirements for amino acid transport and a variety of amino acid transport mechanisms, including the aforementioned families. Further it focuses on the large group of Nutrient Amino acid Transporters (NATs), which comprise a recently identified subfamily of the Neurotransmitter Sodium Symporter family (NSS or SLC6). The first insect NAT, cloned from the caterpillar gut, has a broad substrate spectrum similar to mammalian B(0) transporters. Several new NAT-SLC6 members have been characterized in an effort to explore mechanisms for the essential amino acid absorption in model dipteran insects. The identification and functional characterization of new B(0)-like and narrow specificity transporters of essential amino acids in fruit fly and mosquitoes leads to a fundamentally important insight: that NATs evolved and act together as the integrated active core of a transport network that mediates active alimentary absorption and systemic distribution of essential amino acids. This role of NATs is projected from the most primitive prokaryotes to the most complex metazoan organisms, and represents an interesting platform for unraveling the molecular evolution of amino acid transport and modeling amino acid transport disorders. The comparative study of NATs elucidates important adaptive differences between essential amino acid transportomes of invertebrate and vertebrate organisms, outlining a new possibility for selective targeting of essential amino acid absorption mechanisms to control medically and economically important arthropods and other invertebrate organisms.     

The multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) exporter superfamily.

The multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) exporter superfamily (TC #2.A.66) consists of four previously recognized families: (a) the ubiquitous multi-drug and toxin extrusion (MATE) family; (b) the prokaryotic polysaccharide transporter (PST) family; (c) the eukaryotic oligosaccharidyl-lipid flippase (OLF) family and (d) the bacterial mouse virulence factor family (MVF). Of these four families, only members of the MATE family have been shown to function mechanistically as secondary carriers, and no member of the MVF family has been shown to function as a transporter. Establishment of a common origin for the MATE, PST, OLF and MVF families suggests a common mechanism of action as secondary carriers catalyzing substrate/cation antiport. Most protein members of these four families exhibit 12 putative transmembrane alpha-helical segments (TMSs), and several have been shown to have arisen by an internal gene duplication event; topological variation is observed for some members of the superfamily. The PST family is more closely related to the MATE, OLF and MVF families than any of these latter three families are related to each other. This fact leads to the suggestion that primordial proteins most closely related to the PST family were the evolutionary precursors of all members of the MOP superfamily. Here, phylogenetic trees and average hydropathy, similarity and amphipathicity plots for members of the four families are derived and provide detailed evolutionary and structural information about these proteins. We show that each family exhibits unique characteristics. For example, the MATE and PST families are characterized by numerous paralogues within a single organism (58 paralogues of the MATE family are present in Arabidopsis thaliana), while the OLF family consists exclusively of orthologues, and the MVF family consists primarily of orthologues. Only in the PST family has extensive lateral transfer of the encoding genes occurred, and in this family as well as the MVF family, topological variation is a characteristic feature. The results serve to define a large superfamily of transporters that we predict function to export substrates using a monovalent cation antiport mechanism.     

Phylogenetic relationships within cation transporter families of Arabidopsis

Uptake and translocation of cationic nutrients play essential roles in physiological processes including plant growth, nutrition, signal transduction, and development. Approximately 5% of the Arabidopsis genome appears to encode membrane transport proteins. These proteins are classified in 46 unique families containing approximately 880 members. In addition, several hundred putative transporters have not yet been assigned to families. In this paper, we have analyzed the phylogenetic relationships of over 150 cation transport proteins. This analysis has focused on cation transporter gene families for which initial characterizations have been achieved for individual members, including potassium transporters and channels, sodium transporters, calcium antiporters, cyclic nucleotide-gated channels, cation diffusion facilitator proteins, natural resistance-associated macrophage proteins (NRAMP), and Zn-regulated transporter Fe-regulated transporter-like proteins. Phylogenetic trees of each family define the evolutionary relationships of the members to each other. These families contain numerous members, indicating diverse functions in vivo. Closely related isoforms and separate subfamilies exist within many of these gene families, indicating possible redundancies and specialized functions. To facilitate their further study, the PlantsT database (http://plantst.sdsc.edu) has been created that includes alignments of the analyzed cation transporters and their chromosomal locations.     

Prediction of transporter family from protein sequence by support vector machine approach

Transporters play key roles in cellular transport and metabolic processes, and in facilitating drug delivery and excretion. These proteins are classified into families based on the transporter classification (TC) system. Determination of the TC family of transporters facilitates the study of their cellular and pharmacological functions. Methods for predicting TC family without sequence alignments or clustering are particularly useful for studying novel transporters whose function cannot be determined by sequence similarity. This work explores the use of a machine learning method, support vector machines (SVMs), for predicting the family of transporters from their sequence without the use of sequence similarity. A total of 10,636 transporters in 13 TC subclasses, 1914 transporters in eight TC families, and 168,341 nontransporter proteins are used to train and test the SVM prediction system. Testing results by using a separate set of 4351 transporters and 83,151 nontransporter proteins show that the overall accuracy for predicting members of these TC subclasses and families is 83.4% and 88.0%, respectively, and that of nonmembers is 99.3% and 96.6%, respectively. The accuracies for predicting members and nonmembers of individual TC subclasses are in the range of 70.7-96.1% and 97.6-99.9%, respectively, and those of individual TC families are in the range of 60.6-97.1% and 91.5-99.4%, respectively. A further test by using 26,139 transmembrane proteins outside each of the 13 TC subclasses shows that 90.4-99.6% of these are correctly predicted. Our study suggests that the SVM is potentially useful for facilitating functional study of transporters irrespective of sequence similarity.     

Molecular Mechanism of Nramp-Family Transition Metal Transport

The Natural resistance-associated macrophage protein (Nramp) family of transition metal transporters enables uptake and trafficking of essential micronutrients that all organisms must acquire to survive. Two decades after Nramps were identified as proton-driven, voltage-dependent secondary transporters, multiple Nramp crystal structures have begun to illustrate the fine details of the transport process and provide a new framework for understanding a wealth of preexisting biochemical data. Here we review the relevant literature pertaining to Nramps’ biological roles and especially their conserved molecular mechanism, including our updated understanding of conformational change, metal binding and transport, substrate selectivity, proton transport, proton-metal coupling, and voltage dependence. We ultimately describe how the Nramp family has adapted the LeuT fold common to many secondary transporters to provide selective transition-metal transport with a mechanism that deviates from the canonical model of symport.