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ATP-binding cassette (ABC) transporters, although being ubiquitous in biology, often feature a subunit that is limited primarily to bacteria and archaea. This subunit, the substrate-binding protein (SBP), is a key determinant of the substrate specificity and high affinity of ABC uptake systems in these organisms. Most prokaryotes have many SBP-dependent ABC transporters that recognize a broad range of ligands from metal ions to amino acids, sugars and peptides. Herein, we review the structure and function of a number of more unusual SBPs, including an ABC transporter involved in the transport of rare furanose forms of sugars and an SBP that has evolved to specifically recognize the bacterial cell wall-derived murein tripeptide (Mtp). Both these examples illustrate that subtle changes in binding-site architecture, including changes in side chains not directly involved in ligand co-ordination, can result in significant alteration of substrate range in novel and unpredictable ways.

Monosaccharides available in the extracellular milieu of Agrobacterium tumefaciens can be transported into the cytoplasm, or via the periplasmic sugar binding protein, ChvE, play a critical role in controlling virulence gene expression. The ChvE-MmsAB ABC transporter is involved in the utilization of a wide range of monosaccharide substrates but redundant transporters are likely given the ability of a chvE-mmsAB deletion strain to grow, albeit more slowly, in the presence of particular monosaccharides. In this study, a putative ABC transporter encoded by the gxySBA operon is identified and shown to be involved in the utilization of glucose, xylose, fucose, and arabinose, which are also substrates for the ChvE-MmsAB ABC transporter. Significantly, GxySBA is also shown to be the first characterized glucosamine ABC transporter. The divergently transcribed gene gxyR encodes a repressor of the gxySBA operon, the function of which can be relieved by a subset of the transported sugars, including glucose, xylose, and glucosamine, and this substrate-induced expression can be repressed by glycerol. Furthermore, deletion of the transporter can increase the sensitivity of the virulence gene expression system to certain sugars that regulate it. Collectively, the results reveal a remarkably diverse set of substrates for the GxySBA transporter and its contribution to the repression of sugar sensitivity by the virulence-controlling system, thereby facilitating the capacity of the bacterium to distinguish between the soil and plant environments.

Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding., Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

Type-1 secretion systems (T1SSs) represent a widespread mode of protein secretion across the cell envelope in Gram-negative bacteria. The T1SS is composed of an inner-membrane ABC transporter, a periplasmic membrane-fusion protein, and an outer-membrane porin. These three components assemble into a complex spanning both membranes and providing a conduit for the translocation of unfolded polypeptides. We show that ATP hydrolysis and assembly of the entire T1SS complex is necessary for protein secretion. Furthermore, we present a 3.15-A crystal structure of AaPrtD, the ABC transporter found in the Aquifex aeolicus T1SS. The structure suggests a substrate entry window just above the transporter's nucleotide binding domains. In addition, highly kinked transmembrane helices, which frame a narrow channel not observed in canonical peptide transporters, are likely involved in substrate translocation. Overall, the AaPrtD structure supports a polypeptide transport mechanism distinct from alternating access.

Summary Antimicrobial peptides (AMPs) are a group of antibiotics that mainly target the cell wall of Gram-positive bacteria. Resistance is achieved by a variety of mechanisms including target alterations, changes in the cell's surface charge, expression of immunity peptides or by dedicated ABC transporters. The latter often provide the greatest level of protection. Apart from resistance, ABC transporters are also required for the export of peptides during biosynthesis. In this review the different AMP transporters identified to date in Firmicutes bacteria were classified into five distinct groups based on their domain architecture, two groups with a role in biosynthesis, and three involved in resistance. Comparison of the available information for each group regarding function, transport mechanism and gene regulation revealed distinguishing characteristics as well as common traits. For example, a strong correlation between transporter group and mode of gene regulation was observed, with three different types of two-component systems as well as XRE family transcriptional regulators commonly associated with individual transporter groups. Furthermore, the presented summary of the state-of-the-art on AMP transport in Firmicutes bacteria, discussed in the context of transporter phylogeny, provides insights into the mechanisms of substrate translocation and how this may result in resistance against compounds that bind extracellular targets.

Tripartite ATP-independent periplasmic (TRAP) transporters are widespread in bacteria but poorly characterized. They contain three subunits, a small membrane protein, a large membrane protein, and a substrate-binding protein (SBP). Although the function of the SBP is well established, the membrane components have only been studied in detail for the sialic acid TRAP transporter SiaPQM from Haemophilus influenzae, where the membrane proteins are genetically fused. Herein, we report the first in vitro characterization of a truly tripartite TRAP transporter, the SiaPQM system (VC1777-1779) from the human pathogen Vibrio cholerae. The active reconstituted transporter catalyzes unidirectional Na(+)-dependent sialic acid uptake having similar biochemical features to the orthologous system in H. influenzae. However, using this tripartite transporter, we demonstrate the tight association of the small, SiaQ, and large, SiaM, membrane proteins that form a 1:1 complex. Using reconstituted proteoliposomes containing particular combinations of the three subunits, we demonstrate biochemically that all three subunits are likely to be essential to form a functional TRAP transporter.

Streptococcus mutans , a Gram-positive organism, is the primary causative agent in the formation of dental caries in humans. To persist in the oral cavity, S. mutans must be able to tolerate rapid environmental fluctuations and exposure to various toxic chemicals. However, the mechanisms underlying the ability of this cariogenic pathogen to survive and proliferate under harsh environmental conditions remain largely unknown. Here, we wanted to understand the mechanisms by which S. mutans withstands exposure to methyl viologen (MV), a quaternary ammonium compound (QAC) that generates superoxide radicals in the cell. To elucidate the essential genes for MV tolerance, screening of ∼3,500 mutants generated by IS S1 mutagenesis, revealed 15 MV-sensitive mutants. Among them, five and four independent insertions had occurred in SMU.905 and SMU.906 genes, respectively. These two genes are appeared to be organized in an operon and encode a putative ABC transporter complex; we designated the genes as vltA and vltB , for v io l ogen t ransporter. To verify our results, vltA was deleted by using an antibiotic resistance marker; the mutant was just as sensitive to MV as the IS S1 insertion mutants. Furthermore, vltA and vltB mutants were also sensitive to other viologen compounds such as benzyl and ethyl viologens. Complementation assays were also carried out to confirm the role of VltA and VltB in viologen tolerance. Sensitivity to various drugs, including a wide range of QACs, was evaluated. It appears that a functional VltA is also required for full resistance toward acriflavin, ethidium bromide, and safranin; all are well-known QACs. These results indicate that VltA/B constitute a heterodimeric multidrug efflux pump of the ABC family. BLAST-P analysis suggests that homologs of VltA/B are widely present in streptococci, enterococci, and other important Gram-positive pathogens.

Substrate-binding proteins (SBP) are associated with a wide variety of protein complexes. The proteins are part of ATP-binding cassette transporters for substrate uptake, ion gradient driven transporters, DNA-binding proteins, as well as channels and receptors from both pro-and eukaryotes. A wealth of structural and functional data is available on SBPs, with over 120 unique entries in the Protein Data Bank (PDB). Over a decade ago these proteins were divided into three structural classes, but based on the currently available wealth of structural data, we propose a new classification into six clusters, based on features of their three-dimensional structure. (C) 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Transporters influence the disposition of chemicals within the body by participating in absorption, distribution, and elimination. Transporters of the solute carrier family (SLC) comprise a variety of proteins, including organic cation transporters (OCT) 1 to 3, organic cation/carnitine transporters (OCTN) 1 to 3, organic anion transporters (OAT) 1 to 7, various organic anion transporting polypeptide isoforms, sodium taurocholate cotransporting polypeptide, apical sodium-dependent bile acid transporter, peptide transporters (PEPT) 1 and 2, concentrative nucleoside transporters (CNT) 1 to 3, equilibrative nucleoside transporter (ENT) 1 to 3, and multidrug and toxin extrusion transporters (MATE) 1 and 2, which mediate the uptake (except MATEs) of organic anions and cations as well as peptides and nucleosides. Efflux transporters of the ATP-binding cassette superfamily, such as ATP-binding cassette transporter A1 (ABCA1), multidrug resistance proteins (MDR) 1 and 2, bile salt export pump, multidrug resistance-associated proteins (MRP) 1 to 9, breast cancer resistance protein, and ATP-binding cassette subfamily G members 5 and 8, are responsible for the unidirectional export of endogenous and exogenous substances. Other efflux transporters [ATPase copper-transporting beta polypeptide (ATP7B) and ATPase class I type 8B member 1 (ATP8B1) as well as organic solute transporters (OST) alpha and beta] also play major roles in the transport of some endogenous chemicals across biological membranes. This review article provides a comprehensive overview of these transporters (both rodent and human) with regard to tissue distribution, subcellular localization, and substrate preferences. Because uptake and efflux transporters are expressed in multiple cell types, the roles of transporters in a variety of tissues, including the liver, kidneys, intestine, brain, heart, placenta, mammary glands, immune cells, and testes are discussed. Attention is also placed upon a variety of regulatory factors that influence transporter expression and function, including transcriptional activation and post-translational modifications as well as subcellular trafficking. Sex differences, ontogeny, and pharmacological and toxicological regulation of transporters are also addressed. Transporters are important transmembrane proteins that mediate the cellular entry and exit of a wide range of substrates throughout the body and thereby play important roles in human physiology, pharmacology, pathology, and toxicology.

Acquiring nutrients from the environment is essential for all microbes, and the ATP-binding cassette (ABC) transporters are one of the major routes by which bacteria achieve it. In this issue of Molecular Microbiology, Chen et al. describe their characterization of what appeared at first glance a simple ABC transporter for acquisition of quaternary ammonium compounds (QACs) in Pseudomonas sp., but their persistence in fully determining the properties of this system led to the experimental demonstration that QAC uptake utilizes three different substrate-binding proteins (SBPs), two of which are encoded at remote locations on the genome as 'orphan' SBPs that are each able to function with a single core ABC transporter. Building on the unusual nature of this system, in which multiple SBPs with non-overlapping substrate specificities compete for the same transporter binding site, they designed elegant in vivo experiments that suggest that only substrate-bound SBPs are able to form functional complexes with the membrane domains. This new finding provides an important piece of in vivo data leading to further insight into how this ubiquitous family of transporters operates.

ABC transporters are ubiquitous membrane proteins that translocate solutes across biological membranes at the expense of ATP. In prokaryotic ABC importers, the extracytoplasmic anchoring of the substrate-binding protein (receptor) is emerging as a key determinant for the structural rearrangements in the cytoplasmically exposed ATP-binding cassette domains and in the transmembrane gates during the nucleotide cycle. Here the molecular mechanism of such signaling events was addressed by electron paramagnetic resonance spectroscopy of spin-labeled ATP-binding cassette maltose transporter variants (MalFGK2-E). A series of doubly spin-labeled mutants in the MalF-P2 domain involving positions 92, 205, 239, 252, and 273 and one triple mutant labeled at positions 205/252 in P2 and 83 in the Q-loop of MalK were assayed. The EPR data revealed that the substrate-binding protein MalE is bound to the transporter throughout the transport cycle. Concomitantly with the three conformations of the ATP-binding cassette MalK2, three functionally relevant conformations are found also in the periplasmic MalF-P2 loop, strictly dependent on cytoplasmic nucleotide binding and periplasmic docking of liganded MalE to MalFG. The reciprocal communication across the membrane unveiled here gives first insights into the stimulatory effect of MalE on the ATPase activity, and it is suggested to be an important mechanistic feature of receptor-coupled ABC transporters.

The shuttling of substrates across a cellular membrane frequently requires a specialized ATP-binding cassette (ABC) transporter, which couples the energy of ATP binding and hydrolysis to substrate transport. Due to its importance in immunity, the ABC transporter associated with antigen processing (TAP) has been studied extensively and is an excellent model for other ABC transporters. The TAP protein pumps cytosolic peptides into the endoplasmic reticulum for loading onto class I major histocompatibility complex (MHC) for subsequent immune surveillance. Here, we outline a potential mechanism for the TAP protein with supporting evidence from bacterial transporter structures. The similarities and differences between TAP and other transporters support the notion that ABC transporters in general have adapted around a universal transport mechanism.

Pharmacodynamical studies showed that most drugs elicit their effects by acting on 3 kinds of protein molecules known as receptors, enzymes or transporters. Although their detail properties had not been explained for decades the roles of transporters in drug kinetics and dynamics has been well understood, even have been applied in the therapy. Transporters are classified into 2 major classes, the solute carriers (SLC) and ATP-binding cassette (ABC) families. SLC transporters do not possess ATP binding site property as those of ABC transporters. SLC transporters consist of 3 SLC subfamilies i.e. organic cation transporters (OCTs), organic anion transporters (OATs) and organic anion transporting polypeptides (OATPs). In contrast, ABC transporters require ATP hydrolysis to transport substrate across cell membrane. Human ABC-transporters consist of ABCA1- 13, ABCB1-11, ABCC1-12, ABCD1-4, ABCE1, ABCF1-3 and ABCG1-8 subfamily. Although the originally funtion of transporter is to transport specific physiological substrate such as nutrient, hormone, cytokines, neurotransmitters and other physiological subtances across cell membrane the specificity is not restricted to each substrate. Drugs and other xenobiotics which have structural similarity to the physiological substrates are recognized and transported by the related transporters. The competition of them on transporters therefore may lead to the occurence of drug-drug interactions (DDI) or drugphysiological substrate interaction in the drug-kinetics phase. Many transporters located in the liver, intestinal and renal epithelial cell membranes involve in the transport of endogenous substance or xenobiotics including drugs play important roles as protective barrier. Since transporters also serve as the targets of drug action it is understood that transporters play important role in the pathogenesis of diseases as well as in the drug therapy of diseases.

The sialic acids are a family of 9-carbon sugar acids found predominantly on the cell-surface glycans of humans and other animals within the Deuterostomes and are also used in the biology of a wide range of bacteria that often live in association with these animals. For many bacteria sialic acids are simply a convenient source of food, whereas for some pathogens they are also used in immune evasion strategies. Many bacteria that use sialic acids derive them from the environment and so are dependent on sialic acid uptake. In this mini-review I will describe the discovery and characterization of bacterial sialic acids transporters, revealing that they have evolved multiple times across multiple diverse families of transporters, including the ATP-binding cassette (ABC), tripartite ATP-independent periplasmic (TRAP), major facilitator superfamily (MFS) and sodium solute symporter (SSS) transporter families. In addition there is evidence for protein-mediated transport of sialic acids across the outer membrane of Gram negative bacteria, which can be coupled to periplasmic processing of different sialic acids to the most common form, β-D-N-acetylneuraminic acid (Neu5Ac) that is most frequently taken up into the cell.

The recent solution of the crystal structure of an entire binding-protein-dependent ABC transporter complex from the archaeon Archaeoglobus fulgidus by Locher and his colleagues marks a milestone in the understanding of the ABC transport mechanism. The structure elegantly demonstrates how the motor ATPase alternately opens and closes the inside and outside pores of the transporter and how the substrate-binding protein delivers its substrate. Binding-protein-dependent sugar ABC transporters in the archaea and in bacteria have an additional feature that could connect ABC transporters to gene regulation and to the control of transport activity by cellular processes.

ABC transporters make a large and diverse family of proteins found in all phylae. AtCCMA is the nucleotide binding domain of a novel Arabidopsis mitochondrial ABC transporter. It is encoded in the nucleus and imported into mitochondria. Sub-organellar and topology studies find AtCCMA bound to the mitochondrial inner membrane, facing the matrix. AtCCMA exhibits an ATPase activity, and ATP/Mg2+ can facilitate its dissociation from membranes. Blue Native PAGE shows that it is part of a 480-kDa complex. Yeast two-hybrid assays reveal interactions between AtCCMA and domains of CcmB, the mitochondria-encoded transmembrane protein of a conserved ABC transporter. All these properties designate the protein as the ortholog in plant mitochondria of the bacterial CcmA required for cytochrome c maturation. The transporter that involves AtCCMA defines a new category of eukaryotic ABC proteins because its transmembrane and nucleotide binding domains are encoded by separate genomes.

Tripartite ATP-independent periplasmic (TRAP) transporters are a family of extracytoplasmic solute receptor-dependent secondary transporters that are widespread in the prokaryotic world but which have not been extensively studied. Here, we present results of a genome-wide analysis of TRAP sequences and genome organization from application of TRAPDb, a relational database created for the collection, curation and analysis of TRAP sequences. This has revealed a specific enrichment in the number of TRAP transporters in several bacteria which is consistent with increased use of TRAP transporters in saline environments. Additionally, we report a number of new organizations of TRAP transporter genes and proteins which suggest the recruitment of TRAP transporter components for use in other biological contexts.

Exogenous 3-hydroxybutyrate can be utilized by a variety of soil bacteria as a carbon and energy source. However, the membrane transporter responsible for 3-hydroxybutyrate uptake remains unidentified. The Bacillus subtilis strain 168 gene yxjC (herein renamed hbuT) encodes a putative gluconate transporter GntT-type membrane transporter with a previously unknown function. hbuT is organized within the same operon with genes that are used for metabolism of 3-hydroxybutyrate. Here we report that a null mutation of hbuT reduced uptake of 3-hydroxybutyrate by B. subtilis cells grown in nutrient sporulation medium. The SigE-controlled HbuT transporter apparently plays a major role in the uptake of 3-hydroxybutyrate. Uptake of 3-hydroxybutyrate by the HbuT transporter occurred in a specific manner at the early sporulation stage. SigE-controlled hbuT expression and 3-hydroxybutyrate uptake were also subject to CcpA-mediated glucose repression. hbuT expression was not induced by exogenous 3-hydroxybutyrate and B. subtilis cells could not utilize 3-hydroxybutyrate as a sole carbon source for growth. HbuT homologs are present in a wide variety of Gram-positive Bacillus species, some Gram-negative Acinetobacter species and a small group of other bacteria. This is the first tentative identification of a membrane transporter responsible for the uptake of 3-hydroxybutyrate in bacteria.

ATP binding cassette (ABC) transporters constitute a very large and ubiquitous superfamily of membrane proteins. They are responsible for ATP hydrolysis driven translocation of countless substrates. Being a very old and diverse group of proteins present in all organisms they share a common feature, which is the presence of an evolutionary conservative nucleotide binding domain (NBD)--the engine that drives the transport. Another common domain is a transmembrane domain (TMD) which consists of several membrane-spanning helices. This part of protein is substrate-specific, thus it is much more variable. ABC transporters are known for driving drug efflux in many pathogens and cancer cells, therefore they are the subject of extensive studies. There are many examples of conferring a drug resistance phenotype in fungal pathogens by ABC transporters, however, little is known about these proteins in dermatophytes--a group of fungi causing superficial mycoses. So far only a single ABC transporter has been extensively studied in this group of pathogens. We analyzed available genomic sequences of seven dermatophyte species in order to provide an insight into dermatophyte ABC protein inventory. Phylogenetic studies of ABC transporter genes and their products were conducted and included ABC transporters of other fungi. Our results show that each dermatophyte genome studied possesses a great variety of ABC transporter genes. Detailed analysis of selected genes and their products indicates that relatively recent duplication of ABC transporter genes could lead to novel substrate specificity.

The hydrophobic substrate-selective domain of the resistance-nodulation-division-type xenobiotic transporter of Pseudomonas aeruginosa was assigned based on the different organic-solvent selectivities of MexB and MexY. The MexB-MexY hybrid protein consisting of two large periplasmic domains of MexB and the transmembrane domains of MexY showed MexB-type organic solvent selectivity. The results imply that the resistance-nodulation-division-type xenobiotic transporters recognize hydrophobic substrates such as organic solvents by their periplasmic domains and expel them to the external milieu. This is an elegant way to protect the cytoplasmic membrane from membrane-deteriorating agents.

Tripartite ATP-independent periplasmic (TRAP) transporters are relatively common prokaryotic secondary transporters which comprise an extracytoplasmic solute receptor (ESR) protein and two dissimilar membrane proteins or domains, yet the substrates and physiological functions of only a few of these systems are so far known. In this study, a biophysical approach was used to identify the ligands for the purified Rhodobacter capsulatus RRC01191 and Escherichia coli YiaO proteins, which are members of two phylogenetically distinct families of TRAP-ESRs found in diverse bacteria. In contrast to previous indirect evidence pointing to RRC01191 orthologues being involved in polyol uptake, it was shown that RRC01191 binds pyruvate, 2-oxobutyrate and a broad range of aliphatic monocarboxylic 2-oxoacid anions with varying affinities (K(d) values 0.08-3 muM), consistent with a predicted role in monocarboxylate transport related to branched-chain amino-acid biosynthesis. The E. coli YiaMNO TRAP transporter has previously been proposed to be an l-xylulose uptake system [Plantinga et al. (2004) Mol Membr Biol 21, 51-57], but purified YiaO did not bind l- or d-xylulose as judged by fluorescence spectroscopy, circular dichroism or mass spectrometry. Instead, these techniques showed that a breakdown product of l-ascorbate, 2,3-diketo-l-gulonate (2,3-DKG), binds by a simple one-step mechanism with sub-micromolar affinity. The data provide the first evidence for the existence of ESR-dependent transporters for 2-oxoacids and 2,3-DKG, homologues of which appear to be widespread amongst prokaryotes. The results also underline the utility of direct ESR ligand-binding studies for TRAP transporter characterization.

Many bacteria use an ABC transporter for high-affinity uptake of zinc with a cluster 9 solute-binding protein. Other members of this protein family transport manganese. At present, it is not always possible to distinguish zinc-specific and manganese-specific transporters on the basis of sequence analysis. Low-affinity ZIP-type zinc transporters in bacteria have also been identified. Most high-affinity zinc uptake systems are regulated by Zur proteins, which form at least three unrelated subgroups of the Fur protein family (regulators of iron transport). High-affinity transport of zinc out of the periplasmic space poses a problem to the cell because zinc is a cofactor of several periplasmic enzymes. Certain zinc-binding proteins in the periplasm might function as chaperones to supply these enzymes with zinc.

ATP-binding cassette (ABC) transporters are widespread among living organisms and comprise one of the largest protein families. For example, components of ABC transporters are encoded by approximately 5% of the Escherichia coli and Bacillus subtilis genomes ([47][1], [80][2]). These transporters are

More than 50 distinct amino acid transporter genes have been identified in the genome of Arabidopsis, indicating that transport of amino acids across membranes is a highly complex feature in plants. Based on sequence similarity, these transporters can be divided into two major superfamilies: the amino acid transporter family and the amino acid polyamine choline transporter family. Currently, mainly transporters of the amino acid transporter family have been characterized. Here, a molecular and functional characterization of amino acid polyamine choline transporters is presented, namely the cationic amino acid transporter (CAT) subfamily. CAT5 functions as a high-affinity, basic amino acid transporter at the plasma membrane. Uptake of toxic amino acid analogs implies that neutral or acidic amino acids are preferentially transported by CAT3, CAT6, and CAT8. The expression profiles suggest that CAT5 may function in reuptake of leaking amino acids at the leaf margin, while CAT8 is expressed in young and rapidly dividing tissues such as young leaves and root apical meristem. CAT2 is localized to the tonoplast in transformed Arabidopsis protoplasts and thus may encode the long-sought vacuolar amino acid transporter.

ATP-binding cassette (ABC) transporters are multidomain integral membrane proteins that utilise the energy of ATP hydrolysis to translocate solutes across cellular membranes in all phyla. ABC transporters form one of the largest of all protein families and are central to many important biomedical phenomena, including resistance of cancers and pathogenic microbes to drugs. Elucidation of the structure and mechanism of ABC transporters is essential to the rational design of agents to control their function. While a wealth of high-resolution structures of ABC proteins have been produced in recent years, many fundamental questions regarding the protein's mechanism remain unanswered. In this review, we examine the recent structural data concerning ABC transporters and related proteins in the light of other experimental and theoretical data, and discuss these data in relation to current ideas concerning the transporters' molecular mechanism.

In archaea, ATP-binding cassette (ABC) transporters play a crucial role in substrate uptake, export, and osmoregulation. Archaeal substrate-binding-protein-dependent ABC transporters are equipped with a very high affinity for their cognate substrates which provide these organisms with the ability to efficiently scavenge substrates from their environment even when present only at low concentration. Further adaptations to the archaeal way of life are especially found in the domain organization and anchoring of the substrate-binding proteins to the membrane. Examination of the signal peptides of binding proteins of 14 archaeal genomes showed clear differences between euryarchaeotes and crenarchaeotes. Furthermore, a profiling and comparison of ABC transporters in the three sequenced pyrococcal strains was performed.

The yiaMNO genes of Escherichia coli K-12 encode a binding protein-dependent secondary, or tri-partite ATP-independent periplasmic (TRAP), transporter. Since only a few members of this family have been functionally characterized to date, we aimed to identify the substrate for this transporter. Cells that constitutively express the yiaK-S gene cluster metabolized the rare pentose L-xylulose, while deletion of the yiaMNO transporter genes reduced L-xylulose metabolism. The periplasmic substrate-binding protein YiaO was found to bind L-xylulose, and stimulated L-xylulose uptake by spheroplasts. These data indicate that the yiaMNO transporter mediates uptake of this rare pentose.

Extracytoplasmic solute binding receptors are constituents of primary and secondary active transport systems. Previous studies have shown that the constituents of two such families (ABC and TRAP-T) occur in bacteria and archaea and have undergone minimal shuffling of constituents between systems during evolutionary history. We here show that a third family of binding receptor-dependent transporters, the tripartite tricarboxylate transporter (TTT) family, the prototype of which is the TctABC system of Salmonella typhimurium, occurs in many bacteria but not in archaea or eukaryotes. Phylogenetic analyses suggest that these systems have evolved from a primordial tripartite system with only two out of 39 possible examples of shuffling of constituents between systems. The occurrence of TctA homologues in many bacteria and archaea that apparently lack corresponding TctB and TctC homologues suggests that the appearance of tripartite systems was a relatively recent evolutionary invention that occurred after the divergence of archaea and eukaryotes from bacteria.

The ATPase components of ATP binding cassette (ABC) transporters power the transporters by binding and hydrolyzing ATP. Major conformational changes of an ATPase are revealed by crystal structures of MalK, the ATPase subunit of the maltose transporter from Escherichia coli, in three different dimeric configurations. While other nucleotide binding domains or subunits display low affinity for each other in the absence of the transmembrane segments, the MalK dimer is stabilized through interactions of the additional C-terminal domains. In the two nucleotide-free structures, the N-terminal nucleotide binding domains are separated to differing degrees, and the dimer is maintained through contacts of the C-terminal regulatory domains. In the ATP-bound form, the nucleotide binding domains make contact and two ATPs lie buried along the dimer interface. The two nucleotide binding domains of the dimer open and close like a pair of tweezers, suggesting a regulatory mechanism for ATPase activity that may be tightly coupled to translocation.

Intracellular thiols like L-cystine and L-cystine play a critical role in the regulation of cellular processes. Here we show that Escherichia coli has two L-cystine transporters, the symporter YdjN and the ATP-binding cassette importer FliY-YecSC. These proteins import L-cystine, an oxidized product of L-cystine from the periplasm to the cytoplasm. The symporter YdjN, which is expected to be a new member of the L-cystine regulon, is a low affinity L-cystine transporter (K m = 1.1 μM) that is mainly involved in L-cystine uptake from outside as a nutrient. E. coli has only two L-cystine importers because ΔydjNΔyecS mutant cells are not capable of growing in the minimal medium containing L-cystine as a sole sulfur source. Another protein YecSC is the FliY-dependent L-cystine transporter that functions cooperatively with the L-cystine transporter YdeD, which exports L-cystine as reducing equivalents from the cytoplasm to the periplasm, to prevent E. coli cells from oxidative stress. The exported L-cystine can reduce the periplasmic hydrogen peroxide to water, and then generated L-cystine is imported back into the cytoplasm via the ATP-binding cassette transporter YecSC with a high affinity to L-cystine (K m = 110 nM) in a manner dependent on FliY, the periplasmic L-cystine-binding protein. The double disruption of ydeD and fliY increased cellular levels of lipid peroxides. From these findings, we propose that the hydrogen peroxide-inducible L-cystine/L-cystine shuttle system plays a role of detoxification of hydrogen peroxide before lipid peroxidation occurs, and then might specific prevent damage to membrane lipids.

ATP-binding cassette (ABC) transporters are a major category of membrane-associated bacterial protein structures involved in the transport of a wide range of substrates. Streptococcus pneumoniae has over 60 ABC transporters, most of which are conserved in all strains. Around two-thirds of S. pneumoniae ABC transporters are importers associated with lipoproteins required for the uptake of various substrates including micronutrients, peptides, and sugars. The remaining ABC transporters export quorum-sensing peptides, drugs (including antibiotics), and antimicrobial peptides from the bacterial cytosol. ABC transporter functions are often partially redundant as other ABC and non-ABC transporter systems import the same substrates. Many ABC transporters have important effects on pathogen–host interactions, either by improving bacterial growth in nutrient-restricted conditions, wider effects on bacterial physiology, or by reducing sensitivity to host antimicrobial peptides or to antibiotics. As a consequence, ABC transporters have a fundamental role in S. pneumoniae biology and virulence.

The recent completion of the Escherichia coli genome sequence (Blattner et al., 1997) has permitted an analysis of the complement of genomically encoded ATP-binding cassette (ABC) proteins. A total of 79 ABC proteins makes this the largest paralogous family of proteins in E. coli. These 79 proteins include 97 ABC domains (as some proteins include more than one ABC domain) and are components of 69 independent functional systems (as many systems involve more than one ABC domain). The ABC domains are often, but not exclusively, the energy-generating domains of multicomponent membrane-bound transporters. Thus, 57 of the 69 systems are ABC transporters, of which 44 are periplasmic-binding protein-dependent uptake systems and 13 are presumed exporters. The genes encoding these ABC transporters occupy almost 5% of the genome. Of the 12 systems that are not obviously transport related, the function of only one, the excision repair protein UvrA, is known. A phylogenetic analysis suggests that the majority of ABC proteins can be assigned to 10 subfamilies. Together with statistical and, importantly, biological evidence, this analysis provides insight into the evolution and function of the ABC proteins.

Studies of periplasmic binding protein-dependent transporters date to the 1960s when it was realized that the transport of certain solutes was inhibited following an osmotic shock that released the contents of the periplasm ([8][1], [80][2]). The ATP-binding cassette (ABC) superfamily ([52][3]) was

▪ Abstract The ATP-binding cassette (ABC) transporters are a family of large proteins in membranes and are able to transport a variety of compounds through membranes against steep concentration gradients at the cost of ATP hydrolysis. The available outline of the human genome contains 48 ABC genes; 16 of these have a known function and 14 are associated with a defined human disease. Major physiological functions of ABC transporters include the transport of lipids, bile salts, toxic compounds, and peptides for antigen presentation or other purposes. We review the functions of mammalian ABC transporters, emphasizing biochemical mechanisms and genetic defects. Our overview illustrates the importance of ABC transporters in human physiology, toxicology, pharmacology, and disease. We focus on three topics: (a) ABC transporters transporting drugs (xenotoxins) and drug conjugates. (b) Mammalian secretory epithelia using ABC transporters to excrete a large number of substances, sometimes against a steep concentration gradient. Several inborn errors in liver metabolism are due to mutations in one of the genes for these pumps; these are discussed. (c) A rapidly increasing number of ABC transporters are found to play a role in lipid transport. Defects in each of these transporters are involved in human inborn or acquired diseases.

Primary active transporters, such as ATP-binding cassette (ABC) transporters, directly utilize the free energy released upon the hydrolysis of ATP to pump substrates against a concentration gradient. The fundamental biological role of the ABC is to couple the energy of ATP hydrolysis to an impressively large variety of essential biological phenomena comprising not only transmembrane transport but also several non-transport-related processes such as translation elongation, translation regulation, ribosome biogenesis, and DNA repair. The nature of the substrates handled by ABC transporters is extraordinarily wide, including mono-and oligosaccharides, organic and inorganic ions, amino acids and short peptides, iron-siderophores, metals, polyamine cations, opines, and vitamins. The major finding is that the ABC proteins or domains fall into three main subdivisions or classes: class 1 comprises systems with fused ABC and IM domains, class 2 comprises systems with two duplicated fused ABC domains and no transmembrane domains, and class 3 contains systems with IM and ABC domains carried by independent polypeptide chains. It can be speculated that at least some membrane proteins C (BMPCs) might be involved in the uptake of an as-yet unidentified monosaccharide. Genes encoding the ATPase of ABC systems constitute about 2% of total genes in most Bacteria and Archaea. A closer look at the distribution of ABC systems among the sequenced genomes reveals several interesting features. Efforts are required to experimentally analyze in detail the functions of ABC transporters.

High-affinity uptake into bacterial cells is mediated by a large class of periplasmic binding protein-dependent transport systems, members of the ATP-binding cassette superfamily. In the maltose transport system of Escherichia coli , the periplasmic maltose-binding protein binds its substrate maltose with high affinity and, in addition, stimulates the ATPase activity of the membrane-associated transporter when maltose is present. Vanadate inhibits maltose transport by trapping ADP in one of the two nucleotide-binding sites of the membrane transporter immediately after ATP hydrolysis, consistent with its ability to mimic the transition state of the γ-phosphate of ATP during hydrolysis. Here we report that the maltose-binding protein becomes tightly associated with the membrane transporter in the presence of vanadate and simultaneously loses its high affinity for maltose. These results suggest a general model explaining how ATP hydrolysis is coupled to substrate transport in which a binding protein stimulates the ATPase activity of its cognate transporter by stabilizing the transition state.

In a soil isolate, Sphingomonas sp. A1, the transport of a macromolecule (alginate: 27 kDa) is mediated by a pit-dependent ATP-binding cassette (ABC) transporter. The transporter is different from other ABC transporters so far analyzed in that its function is dependent on the pit, a mouth-like organ formed on the cell surface only when the cells are compelled to assimilate macromolecules, and in that it allows direct import of macromolecules into cells. The ABC transporter coupled with the pit, which functions as a funnel and/or concentrator of macromolecules to be imported, was designated as the "Super-channel", and in this review, we discuss the three-dimensional structure and specific function of the "Super-channel" for macromolecule import found for the first time in a bacterium.

Zn2+ homeostasis in bacteria is achieved by export systems and uptake systems which are separately regulated by their own regulators. Three types of Zn2+ export systems that protect cells from high toxic concentrations of Zn2+ have been identified: RND multi-drug efflux transporters, P-type ATPases, and cation-diffusion facilitators. The RND type exporters for Zn2+ are only found in a few gram-negative bacteria; they allow a very efficient export across the cytoplasmic membrane and the outer membrane of the cell. P-type ATPases and cation-diffusion facilitators belong to protein families that are also found in eukaryotes. The exporters are regulated in bacteria by MerR-like repressor/activators or by ArsR-like repressors. For the high-affinity uptake of Zn2+, several bindingprotein-dependent ABC transporters belonging to one class have been identified in different bacteria. Zn2+ ABC transporters are regulated by Zur repressors, which belong to the Fur protein family of iron regulators. Little is known about low-affinity Zn2+ uptake under zinc-replete conditions. One known example is the phosphate uptake system Pit, which may cotransport Zn2+ in Escherichia coli. Similarly, the citrate—metal cotransporter CitM in Bacillus subtilis may help to supply Zn2+.

ATP-binding cassette (ABC) systems, also called traffic ATPases, are found in eukaryotes and prokaryotes and almost all participate in the transport of a wide variety of molecules. ABC systems are characterized by a highly conserved ATPase module called here the ABC module, involved in coupling transport to ATP hydrolysis. We have used the sequence of one of the first representatives of bacterial ABC transporters, the MalK protein, to collect 250 closely related sequences from a nonredundant protein sequence database. The sequences collected by this objective method are all known or putative ABC transporters. After having eliminated short protein sequences and duplicates, the 197 remaining sequences were subjected to a phylogenetic analysis based on a mutational similarity matrix. An unrooted tree for these modules was found to display two major branches, one grouping all collected uptake systems and the other all collected export systems. This remarkable disposition strongly suggests that the divergence between these two functionally different types of ABC systems occurred once in the history of these systems and probably before the differentiation of prokaryotes and eukaryotes. We discuss the implications of this finding and we propose a model accounting for the generation and the diversification of ABC systems.

ABC transporters (also known as traffic ATPases) form a large family of proteins responsible for the translocation of a variety of compounds across membranes of both prokaryotes and eukaryotes. The recently completed Escherichia coli genome sequence revealed that the largest family of paralogous E. coli proteins is composed of ABC transporters. Many eukaryotic proteins of medical significance belong to this family, such as the cystic fibrosis transmembrane conductance regulator (CFTR), the P-glycoprotein (or multidrug-resistance protein) and the heterodimeric transporter associated with antigen processing (Tap1-Tap2). Here we report the crystal structure at 1.5 A resolution of HisP, the ATP-binding subunit of the histidine permease, which is an ABC transporter from Salmonella typhimurium. We correlate the details of this structure with the biochemical, genetic and biophysical properties of the wild-type and several mutant HisP proteins. The structure provides a basis for understanding properties of ABC transporters and of defective CFTR proteins.

The ABC transporter is a major class of cellular translocation machinery in all bacterial species encoded in the largest set of paralogous genes. The operon structure is frequently found for the genes of three molecular components: the ATP-binding protein, the membrane protein, and the substrate-binding protein. Here, we developed an “ortholog group table” by comparison and classification of known and putative ABC transporters in the complete genomes of seven microorganisms. Our procedure was to first search and classify the most conserved ATP-binding protein components by the sequence similarity and then to classify the entire transporter units by examining the similarity of the other components and the conservation of the operon structure. The resulting 25 ortholog groups of ABC transporters were well correlated with known functions. Through the analysis, we could assign substrate specificity to hypothetical transporters, predict additional transporter operons, and identify novel types of putative transporters. The ortholog group table was also used as a reference data set for functional assignment in four additional genomes. In general, the ABC transporter operons were strongly conserved despite the extensive shuffling of gene locations in bacterial evolution. InSynechocystis, however, the tendency of forming operons was clearly diminished. Our result suggests that the ancestral ABC transporter operons may have arisen early in evolution before the speciation of bacteria and archaea.

The cytoplasmic membrane proteins of bacterial binding protein‐dependent transporters belong to the superfamily of ABC transporters. The hydrophobic proteins display a conserved, at least 20 amino acid EAA‐‐‐G‐‐‐‐‐‐‐‐‐I‐LP region exposed in the cytosol, the EAA region. We mutagenized the EAA regions of MalF and MalG proteins of the Escherichia coli maltose transport system. Substitutions at the same positions in MalF and MalG have different phenotypes, indicating that EAA regions do not act symmetrically. Mutations in malG or malF that slightly affect or do not affect transport, determine a completely defective phenotype when present together. This suggests that EAA regions of MalF and MalG may interact during transport. Maltose‐negative mutants fall into two categories with respect to the cellular localization of the MalK ATPase: in the first, MalK is membrane‐bound, as in wild‐type strains, while in the second, it is cytosolic, as in strains deleted in the malF and malG genes. From maltose‐negative mutants of the two categories, we isolated suppressor mutations within malK that restore transport. They map mainly in the putative helical domain of MalK, suggesting that EAA regions may constitute a recognition site for the ABC ATPase helical domain.

Transporters are expressed on plasma membranes as well as on intracellular organelle membranes. Transporting solutes across biological membrane is one of the most critical processes for living cells and is essential for all organisms. Correct transporter functioning is essential for maintaining cells and body homeostasis. The discovery of disease-related transporter genes can open new biological pathways and suggest new targets for intervention. Studying the effect of membrane transporters on human diseases is important for understanding drug-related organ toxicity.

P-glycoprotein is a plasma-membrane glycoprotein which confers multidrug-resistance on cells and displays ATP-driven drug-pumping in vitro. It contains two nucleotide-binding domains, and its structure places it in the ‘ABC transporter’ family. We review recent evidence that both nucleotide-sites bind and hydrolyse Mg-ATP. The two catalytic sites interact strongly. A minimal scheme for the MgATP hydrolysis reaction is presented. An alternating catalytic sites scheme is proposed, in which drug transport is coupled to relaxation of a high-energy catalytic site conformation generated by the hydrolysis step. Other ABC transporters may show similar catalytic features.

Thyroid hormones (TH) are essential for the development of the human brain, growth and cellular metabolism. Investigation of TH transporters became one of the emerging fields in thyroid research after the discovery of inactivating mutations in the Monocarboxylate transporter 8 (MCT8), which was found to be highly specific for TH transport. However, additional transmembrane transporters are also very important for TH uptake and efflux in different cell types. They transport TH as secondary substrates and include the aromatic amino acid transporting MCT10, the organic anion transporting polypeptides (e.g. OATP1C1, OATP1A2, OPTP1A4) and the large neutral amino acid transporters (LAT1 and LAT2). These TH transporters characteristically possess 12 transmembrane spanners but due to the strong differing sequences between the three transporter families we assume an identical conformation is not very likely. In contrast to the others, the LAT family members form a heterodimer with the escort protein 4F2hc/CD98. A comparison of sequence proportions, locations and types of functional sensitive features for TH transport discovered by mutations, revealed that transport sensitive charged residues occur as conserved amino acids only within each family of the transporter types but not in all putative TH transporters. Based on the lack of highly conserved sensitive charged residues throughout the three transporter families as a common counterpart for the amino acid moiety of the substrates, we conclude that the molecular transport mechanism is likely organized either a) by different molecular determinants in the divergent transporter types or b) the counterparts for the substrates` amino acid moiety at the transporter are not any charged side chains but other proton acceptors or donators. However, positions of transport sensitive residues coincide at transmembrane helix 8 in the TH transporter MCT8, OATP1C1 and another amino acid transporter, the L-cystine and L-glutamate exchanger xCT, which is highly homologous to LAT1 and LAT2. Here we review the data available and compare similarities and differences between these primary and secondary TH transporters regarding sequences, topology, potential structures, trafficking to the plasma membrane, molecular features and locations of transport sensitive functionalities. Thereby, we focus on TH transporters occurring in the blood-brain barrier.

In Firmicutes bacteria, ATP-binding cassette (ABC) transporters have been recognized as important resistance determinants against antimicrobial peptides. Together with neighboring two-component systems (TCSs), which regulate their expression, they form specific detoxification modules. Both the transport permease and sensor kinase components show unusual domain architecture: the permeases contain a large extracellular domain, while the sensor kinases lack an obvious input domain. One of the best-characterized examples is the bacitracin resistance module BceRS-BceAB of Bacillus subtilis . Strikingly, in this system, the ABC transporter and TCS have an absolute mutual requirement for each other in both sensing of and resistance to bacitracin, suggesting a novel mode of signal transduction in which the transporter constitutes the actual sensor. We identified over 250 such BceAB-like ABC transporters in the current databases. They occurred almost exclusively in Firmicutes bacteria, and 80% of the transporters were associated with a BceRS-like TCS. Phylogenetic analyses of the permease and sensor kinase components revealed a tight evolutionary correlation. Our findings suggest a direct regulatory interaction between the ABC transporters and TCSs, mediating communication between both components. Based on their observed coclustering and conservation of response regulator binding sites, we could identify putative corresponding two-component systems for transporters lacking a regulatory system in their immediate neighborhood. Taken together, our results show that these types of ABC transporters and TCSs have coevolved to form self-sufficient detoxification modules against antimicrobial peptides, widely distributed among Firmicutes bacteria.