ABC transporters: bacterial exporters.

The ABC transporters (also called traffic ATPases) make up a large superfamily of proteins which share a common function and a common ATP-binding domain. ABC transporters are classified into three major groups: bacterial importers (the periplasmic permeases), eukaryotic transporters, and bacterial exporters. We present a comprehensive review of the bacterial ABC exporter group, which currently includes over 40 systems. The bacterial ABC exporter systems are functionally subdivided on the basis of the type of substrate that each translocates. We describe three main groups: protein exporters, peptide exporters, and systems that transport nonprotein substrates. Prototype exporters from each group are described in detail to illustrate our current understanding of this protein family. The prototype systems include the alpha-hemolysin, colicin V, and capsular polysaccharide exporters from Escherichia coli, the protease exporter from Erwinia chrysanthemi, and the glucan exporters from Agrobacterium tumefaciens and Rhizobium meliloti. Phylogenetic analysis of the ATP-binding domains from 29 bacterial ABC exporters indicates that the bacterial ABC exporters can be divided into two primary branches. One branch contains the transport systems where the ATP-binding domain and the membrane-spanning domain are present on the same polypeptide, and the other branch contains the systems where these domains are found on separate polypeptides. Differences in substrate specificity do not correlate with evolutionary relatedness. A complete survey of the known and putative bacterial ABC exporters is included at the end of the review.     

The structures of MsbA: Insight into ABC transporter-mediated multidrug efflux

ATP-binding cassette (ABC) transporters are integral membrane proteins that couple ATP hydrolysis to the transport of various molecules across cellular membranes. Found in both prokaryotes and eukaryotes, a sub-group of these transporters are involved in the efflux of hydrophobic drugs and lipids, causing anti-microbial and chemotherapeutic multidrug resistance. In this review, we examine recent structural and functional analysis of the ABC transporter MsbA and implications on the mechanism of multidrug efflux.     

Functions of ABC transporters in plants

ABC (ATP-binding cassette) proteins are ubiquitously found in prokaryotes and eukaryotes and generally serve as membrane-intrinsic primary active pumps. In higher plants, ABC proteins constitute a large family, grouped phylogenetically into eight clusters, subfamilies ABCA-ABCI (ABCH is not found in plants). ABC transporters shuttle substrates as diverse as lipids, phytohormones, carboxylates, heavy metals, chlorophyll catabolites and xenobiotic conjugates across a variety of biological membranes. To date, the largest proportions of characterized members have been localized to the plasma membrane and the tonoplast, with dominant implications in cellular secretion and vacuolar sequestration, but they are also found in mitochondrial, plastidal and peroxisomal membranes. Originally identified as tonoplast-intrinsic proteins that shuttle xenobiotic conjugates from the cytosol into the vacuole, thus being an integral part of the detoxification machinery, ABC transporters are now recognized to participate in a multitude of physiological processes that allow the plant to adapt to changing environments and cope with biotic and abiotic stresses.     

Bacterial multidrug resistance mediated by a homologue of the human multidrug transporter P-glycoprotein

Most ATP-binding cassette (ABC) multidrug transporters known to date are of eukaryotic origin, such as the P-glycoproteins (Pgps) and multidrug resistance-associated proteins (MRPs). Only one well-characterized ABC multidrug transporter, LmrA, is of bacterial origin. On the basis of its structural and functional characteristics, this bacterial protein is classified as a member of the P-glycoprotein cluster of the ABC transporter superfamily. LmrA can even substitute for P-glycoprotein in human lung fibroblast cells, suggesting that this type of transporter is conserved from bacteria to man. The functional similarity between bacterial LmrA and human P-glycoprotein is further exemplified by their currently known spectrum of substrates, consisting mainly of hydrophobic cationic compounds. In addition, LmrA was found to confer resistance to eight classes of broad-spectrum antibiotics, and homologs of LmrA have been found in pathogenic bacteria, supporting the clinical and academic value of studying this bacterial protein. Current studies are focused on unraveling the mechanism by which ABC multidrug transporters, such as LmrA, couple the hydrolysis of ATP to the translocation of drugs across the membrane. Recent evidence indicates that LmrA mediates drug transport by an alternating two-site transport mechanism.     

Getting In or Out: Early Segregation Between Importers and Exporters in the Evolution of ATP-Binding Cassette (ABC) Transporters

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. Received: 23 February 1997 / Accepted: 7 April 1998     

MsbA ATP-binding cassette (ABC) transporter of E. coli: structure and possible flippase mechanism

ATP-binding cassette (ABC) transporters utilize the energy present in cellular ATP to drive the translocation of structurally diverse set of solutes across the membrane barriers of eubacteria, archaebacteria and eukaryotes. In bacteria, these transporters are considered to be important virulence factors because they play role in nutrient uptake and in the secretion of toxins. The advances in structural determination and functional analysis of bacterial transporters have greatly increased our understanding of the mechanism of transport of these ABC transporters. Although progress in the field of structural biology has been made with the prokaryotic family members, it is likely that eukaryotic transporters will utilize the same mechanisms for translocation process. In this review, we summarize the function of the known MsbA ABC transporters in E. coli and mechanistic insights from structural and possible flippase mechanism studies.     

Evolution of the ATP-binding-cassette transmembrane transporters of vertebrates

The ATP-binding-cassette transmembrane transporters (ABC transporters) known from vertebrates belong to four major subfamilies: (1) the P- glycoproteins (Pgp); (2) the cystic fibrosis transmembrane conductance regulators (CFTR); (3) the Tap proteins encoded with the major histocompatibility complex of mammals; and (4) the peroxisomal membrane proteins. Both Pgp and CFTR have a structure suggesting a past internal gene duplication; a phylogenetic analysis indicated that these duplications occurred independently, while an independent tandem gene duplication occurred in the case of the Tap family. Both the Pgp and Tap proteins show evidence of relationship to bacterial ABC transporters lacking internal duplication, and both are significantly more closely related to the HlyB and MsbA families of transporters from purple bacteria than they are to ABC transporters from nonpurple bacteria. The simplest hypothesis to explain this observation is that eukaryotic Pgp and Tap genes are descended from a mitochondrial gene or genes that were subsequently translocated to the nuclear genome. The Pgp genes of eukaryotes are characterized by a remarkable degree of convergent evolution between the ATP-binding cassettes of their N- terminal and C-terminal halves, whereas no such convergence is seen between the two halves of CFTR genes or between the duplicated Tap genes. Exon 13 of the CFTR gene, which encodes a putative regulatory domain not found in other ABC transporters apart from CFTR, showed high levels of both synonymous and nonsynonymous difference in comparisons among different mammalian species, suggesting that this region is a mutational hot spot.      

WhatEntamoeba histolytica andGiardia lamblia tell us about the evolution of eukaryotic diversity

Entamoeba histolytica andGiardia lamblia are microaerophilic protists, which have long been considered models of ancient pre-mitochondriate eukaryotes. As transitional eukaryotes, amoebae and giardia appeared to lack organelles of higher eukaryotes and to depend upon energy metabolism appropriate for anaerobic conditions early in the history of the planet. However, our studies have shown that amoebae and giardia contain splicoeosomal introns, ras-family signal-transduction proteins, ATP-binding casettes (ABC)-family drug transporters, Golgi, and a mitochondrion-derived organelle (amoebae only). These results suggest that most of the organelles of higher eukaryotes were present in the common ancestor of all eukaryotes, and so dispute the notion of transitional eukaryotic forms. In addition, phylogenetic studies suggest many of the genes encoding the fermentation enzymes of amoebae and giardia derive from prokaryotes by lateral gene transfer (LGT). While LGT has recently been shown to be an important determinant of prokaryotic evolution, this is the first time that LGT has been shown to be an important determinant of eukaryotic evolution. Further, amoebae contain cyst wall-associated lectins, which resemble, but are distinct from lectins in the walls of insects (convergent evolution). Giardia have a novel microtubule-associated structure which tethers together pairs of nuclei during cell division. It appears then that amoebae and giardia tell us less about the origins of eukaryotes and more about the origins of eukaryotic diversity.     

Frequent,independent transfers of a catabolic gene from bacteria to contrasted filamentous eukaryotes

Even genetically distant prokaryotes can exchange genes between them, and these horizontal gene transfer events play a central role in adaptation and evolution. While this was long thought to be restricted to prokaryotes, certain eukaryotes have acquired genes of bacterial origin. However, gene acquisitions in eukaryotes are thought to be much less important in magnitude than in prokaryotes. Here, we describe the complex evolutionary history of a bacterial catabolic gene that has been transferred repeatedly from different bacterial phyla to stramenopiles and fungi. Indeed, phylogenomic analysis pointed to multiple acquisitions of the gene in these filamentous eukaryotes—as many as 15 different events for 65 microeukaryotes. Furthermore, once transferred, this gene acquired introns and was found expressed in mRNA databases for most recipients. Our results show that effective inter-domain transfers and subsequent adaptation of a prokaryotic gene in eukaryotic cells can happen at an unprecedented magnitude.     

Evidence for an early prokaryotic origin of histones H2A and H4 prior to the emergence of eukaryotes.

Histones have been identified recently in many prokaryotes. These histones, unlike their eukaryotic homologs, are of a single uniform type that is thought to resemble the archetypal ancestor of the eukaryotic histone family. In this paper we report the finding, the cloning and the phylogenetic analysis of the sequence of a prokaryotic histone from the hyperthermophile Methanopyrus kandleri . Unlike previously described prokaryotic histones, the Methanopyrus sequence has a novel structure consisting of two tandemly repeated histone fold motifs in a single polypeptide. Sequence analyses indicate that the N-terminal repeat is most closely related to eukaryotic H2A and H4 histones, whereas the C-terminal repeat resembles that found in prokaryotic histones. These results imply an early divergence within the histone gene family prior to the emergence of eukaryotes and may represent an evolutionary step leading to eukaryotic histones.     

Structure and association of ATP-binding cassette transporter nucleotide-binding domains

ATP-binding cassette transporters are responsible for the uptake and efflux of a multitude of substances across both eukaryotic and prokaryotic membranes. Members of this family of proteins are involved in diverse physiological processes including antigen presentation, drug efflux from cancer cells, bacterial nutrient uptake and cystic fibrosis. In order to understand more completely the role of these multidomain transporters an integrated approach combining structural, pharmacological and biochemical methods is being adopted. Recent structural data have been obtained on the cytoplasmic, nucleotide-binding domains of prokaryotic ABC transporters. This review evaluates both these data and the conflicting implications they have for domain communication in ABC transporters. Areas of biochemical research that attempt to resolve these conflicts will be discussed.     

Estimation of phylogenetic inconsistencies in the three domains of life

Discrepancies in phylogenetic trees of bacteria and archaea are often explained as lateral gene transfer events. However, such discrepancies may also be due to phylogenetic artifacts or orthology assignment problems. A first step that may help to resolve this dilemma is to estimate the extent of phylogenetic inconsistencies in trees of prokaryotes in comparison with those of higher eukaryotes, where no lateral gene transfer is expected. To test this, we used 21 proteomes each of eukaryotes (mainly opisthokonts), proteobacteria, and archaea that spanned equivalent levels of genetic divergence. In each domain of life, we defined a set of putative orthologous sequences using a phylogenetic-based orthology protocol and, as a reference topology, we used a tree constructed with concatenated genes of each domain. Our results show, for most of the tests performed, that the magnitude of topological inconsistencies with respect to the reference tree was very similar in the trees of proteobacteria and eukaryotes. When clade support was taken into account, prokaryotes showed some more inconsistencies, but then all values were very low. Discrepancies were only consistently higher in archaea but, as shown by simulation analysis, this is likely due to the particular tree of the archaeal species used here being more difficult to reconstruct, whereas the trees of proteobacteria and eukaryotes were of similar difficulty. Although these results are based on a relatively small number of genes, it seems that phylogenetic reconstruction problems, including orthology assignment problems, have a similar overall effect over prokaryotic and eukaryotic trees based on single genes. Consequently, lateral gene transfer between distant prokaryotic species may have been more rare than previously thought, which opens the way to obtain the tree of life of bacterial and archaeal species using genomic data and the concatenation of adequate genes, in the same way as it is usually done in eukaryotes.     

Genome-Wide Identification,Characterization and Phylogenetic Analysis of ATP-Binding Cassette (ABC) Transporter Genes in Common Carp (Cyprinus carpio)

The ATP-binding cassette (ABC) gene family is considered to be one of the largest gene families in all forms of prokaryotic and eukaryotic life. Although the ABC transporter genes have been annotated in some species, detailed information about the ABC superfamily and the evolutionary characterization of ABC genes in common carp (Cyprinus carpio) are still unclear. In this research, we identified 61 ABC transporter genes in the common carp genome. Phylogenetic analysis revealed that they could be classified into seven subfamilies, namely 11 ABCAs, six ABCBs, 19 ABCCs, eight ABCDs, two ABCEs, four ABCFs, and 11 ABCGs. Comparative analysis of the ABC genes in seven vertebrate species including common carp, showed that at least 10 common carp genes were retained from the third round of whole genome duplication, while 12 duplicated ABC genes may have come from the fourth round of whole genome duplication. Gene losses were also observed for 14 ABC genes. Expression profiles of the 61 ABC genes in six common carp tissues (brain, heart, spleen, kidney, intestine, and gill) revealed extensive functional divergence among the ABC genes. Different copies of some genes had tissue-specific expression patterns, which may indicate some gene function specialization. This study provides essential genomic resources for future studies in common carp.     

Comparative analysis of sequences encoding ABC systems in the genome of the microsporidian Encephalitozoon cuniculi

Microsporidia are amitochondriate eukaryotic microbes with fungal affinities and a common status of obligate intracellular parasites. A set of 13 potential genes encoding ATP-binding cassette (ABC) systems was identified in the fully sequenced genome of Encephalitozoon cuniculi. Our analyses of multiple alignments, phylogenetic trees and conserved motifs support a distribution of E. cuniculi ABC systems within only four subfamilies. Six half transporters are homologous to the yeast ATM1 mitochondrial protein, a finding which is in agreement with the hypothesis of a cryptic mitochondrion-derived compartment playing a role in the synthesis and transport of Fe-S clusters. Five half transporters are similar to the human ABCG1 and ABCG2 proteins, involved in regulation of lipid trafficking and anthracyclin resistance respectively. Two proteins with duplicated ABC domains are clearly candidate to non-transport ABC systems: the first is homologous to mammalian RNase L inhibitor and the second to the yeast translation initiation regulator GCN20. An unusual feature of ABC systems in E. cuniculi is the lack of homologs of P-glycoprotein and other ABC transporters which are involved in multiple drug resistance in a large number of eukaryotic microorganisms.     

Complete inventory of ABC proteins in human pathogenic yeast, Candida albicans

The recent completion of the sequencing project of the opportunistic human pathogenic yeast, Candida albicans (http://www.ncbi.nlm.nih.gov/), led us to analyze and classify its ATP-binding cassette (ABC) proteins, which constitute one of the largest superfamilies of proteins. Some of its members are multidrug transporters responsible for the commonly encountered problem of antifungal resistance. TBLASTN searches together with domain analysis identified 81 nucleotide-binding domains, which belong to 51 different putative open reading frames. Considering that each allelic pair represents a single ABC protein of the Candida genome, the total number of putative members of this superfamily is 28. Domain organization, sequence-based analysis and self-organizing map-based clustering led to the classification of Candida ABC proteins into 6 distinct subfamilies. Each subfamily from C. albicans has an equivalent in Saccharomyces cerevisiae suggesting a close evolutionary relationship between the two yeasts. Our searches also led to the identification of a new motif to each subfamily in Candida that could be used to identify sequences from the corresponding subfamily in other organisms. It is hoped that the inventory of Candida ABC transporters thus created will provide new insights into the role of ABC proteins in antifungal resistance as well as help in the functional characterization of the superfamily of these proteins.     

Mdr65 decreases toxicity of multiple insecticides in Drosophila melanogaster

ABC transporters are ubiquitous membrane-bound proteins, present in both prokaryotes and eukaryotes. The major function of eukaryotic ABC transporters is to mediate the efflux of a variety of substrates (including xenobiotics) out of cells. ABC transporters have been widely investigated in humans, particularly for their involvement in multidrug resistance (MDR). Considerably less is known about their roles in transport and/or excretion in insects. ABC transporters are only known to function as exporters in insects. Drosophila melanogaster has 56 ABC transporter genes, including eight which are phylogenetically most similar to the human Mdr genes (ABCB1 clade). We investigated the role of ABC transporters in the ABCB1 clade in modulating the susceptibility to insecticides. We took advantage of the GAL4/UAS system in D. melanogaster to knockdown the expression levels of Mdr65, Mdr50, Mdr49 and ABCB6 using transgenic UAS-RNAi lines and conditional driver lines. The most notable effects were increased sensitivities to nine different insecticides by silencing of Mdr65. Furthermore, a null mutation of Mdr65 decreased the malathion, malaoxon and fipronil LC₅₀ values by a factor of 1.9, 2.1 and 3.9, respectively. Altogether, this data demonstrates the critical role of ABC transporters, particularly Mdr65, in altering the toxicity of specific, structurally diverse, insecticides in D. melanogaster.     

Phylogenetic analysis of the triterpene cyclase protein family in prokaryotes and eukaryotes suggests bidirectional lateral gene transfer

Functional constraints to modifications in triterpene cyclase amino acid sequences make them good candidates for evolutionary studies on the phylogenetic relatedness of these enzymes in prokaryotes as well as in eukaryotes. In this study, we used a set of identified triterpene cyclases, a group of mainly bacterial squalene cyclases and a group of predominantly eukaryotic oxidosqualene cyclases, as seed sequences to identify 5288 putative triterpene cyclase homologues in publicly available databases. The Cluster Analysis of Sequences software was used to detect groups of sequences with increased pairwise sequence similarity. The sequences fall into two main clusters, a bacterial and a eukaryotic. The conserved, informative regions of a multiple sequence alignment of the family were used to construct a neighbour-joining phylogenetic tree using the AsaturA and maximum likelihood phylogenetic tree using the PhyML software. Both analyses showed that most of the triterpene cyclase sequences were similarly grouped to the accepted taxonomic relationships of the organism the sequences originated from, supporting the idea of vertical transfer of cyclase genes from parent to offspring as the main evolutionary driving force in this protein family. However, a small group of sequences from three bacterial species ( Stigmatella , Gemmata and Methylococcus ) grouped with an otherwise purely eukaryotic cluster of oxidosqualene cyclases, while a small group of sequences from seven fungal species and a sequence from the fern Adiantum grouped consistently with a cluster of otherwise purely bacterial squalene cyclases. This suggests that lateral gene transfer may have taken place, entailing a transfer of oxidosqualene cyclases from eukaryotes to bacteria and a transfer of squalene cyclase from bacteria to an ancestor of the group of Pezizomycotina fungi.     

ATP-binding cassette transporters as pitfalls in selection of transgenic cells

Puromycin, hygromycin, and geneticin (G418) are antibiotics frequently used to select genetically engineered eukaryotic cells after transfection or transduction. Because intrinsic or acquired high expression of ATP-binding cassette (ABC) transporters, such as P-glycoprotein (Pgp/ABCB1) and multidrug resistance-associated proteins (MRP/ABCC1), can hamper efficient selection, it is important to know whether these antibiotics are substrates and/or inducers of efflux transporters. Therefore, we investigated the influence of these antibiotics on drug transporter expression by quantitative real-time polymerase chain reaction in the induction model cell line LS180. Moreover, we assessed whether ABC transporters influence the growth inhibitory effects of these antibiotics by proliferation assays using Madin-Darby canine kidney II (MDCKII) cells overexpressing the particular transporter. The results obtained indicate that puromycin and G418 are substrates of several ABC transporters, mainly Pgp/ABCB1. In contrast, hygromycin seems to be no good substrate for any of the ABC transporters investigated. Puromycin induced ABCC1/MRP1, whereas G418 suppressed ABCB1/Pgp, at the messenger RNA (mRNA) level. In contrast, hygromycin had no effect on ABC transporter mRNA expressions. In conclusion, this study emphasizes the significance of ABC transporters for the efficacy of selection processes. Consciousness of the results is supposed to guide the molecular biologist to the right choice of adequate experimental conditions for successful selection of genetically engineered eukaryotic cells.     

ATP binding cassette importers in eukaryotic organisms

ATP-binding cassette (ABC) transporters are ubiquitous across all realms of life. Dogma suggests that bacterial ABC transporters include both importers and exporters, whilst eukaryotic members of this family are solely exporters, implying that ABC import function was lost during evolution. This view is being challenged, for example energy-coupling factor (ECF)-type ABC importers appear to fulfil important roles in both algae and plants where they form the ABCI sub-family. Herein we discuss whether bacterial Type I and Type II ABC importers also made the transition into extant eukaryotes. Various studies suggest that Type I importers exist in algae and the liverwort family of primitive non-vascular plants, but not in higher plants. The existence of eukaryotic Type II importers is also supported: a transmembrane protein homologous to vitamin B12 import system transmembrane protein (BtuC), hemin transport system transmembrane protein (HmuU) and high-affinity zinc uptake system membrane protein (ZnuB) is present in the Cyanophora paradoxa genome. This protein has homologs within the genomes of red algae. Furthermore, its candidate nucleotide-binding domain (NBD) shows closest similarity to other bacterial Type II importer NBDs such as BtuD. Functional studies suggest that Type I importers have roles in maintaining sulphate levels in the chloroplast, whilst Type II importers probably act as importers of Mn²⁺ or Zn²⁺, as inferred by comparisons with bacterial homologs. Possible explanations for the presence of these transporters in simple plants, but not in other eukaryotic organisms, are considered. In order to utilise the existing nomenclature for eukaryotic ABC proteins, we propose that eukaryotic Type I and II importers be classified as ABCJ and ABCK transporters, respectively.     

Protein exporter function and in vitro ATPase activity are correlated in ABC-domain mutants of HlyB

The Escherichia coli toxin exporter HlyB comprises an integral membrane domain fused to a cytoplasmic domain of the ATP-binding casette (ABC) super-family, and it directs translocation of the 110kDa haemolysin protein out of the bacterial cell without using an N-terminal secretion signal peptide. We have exploited the ability to purify the soluble HlyB ABC domain as a fusion with glutathione S-transferase to obtain a direct correlation of the in vivo export of protein by HlyB with the degree of ATP binding and hydrolysis measured in vitro. Mutations in residues that are invariant or highly conserved in the ATP-binding fold and glycine-rich linker peptide of prokaryotic and eukaryotic ABC transporters caused a complete less of both HlyB exporter function and ATPase activity in proteins still able to bind ATP effectively and undergo ATP-induced conformational change. Mutation of less-conserved residues caused reduced export and ATP hydrolysis, but not ATP binding, whereas substitutions of poorly conserved residues did not impair activity either in vivo or in vitro. The data show that protein export by HlyB has an absolute requirement for the hydrolysis of ATP bound by its cytoplasmic domain and indicate that comparable mutations that disable other prokaryotic and eukaryotic ABC transporters also cause a specific loss of enzymatic activity.