Structural and mechanistic diversity of secondary transporters

Recent reports on the three-dimensional structure of secondary transporters have dramatically increased our knowledge of the translocation mechanism of ions and solutes. The structures of five transporters at atomic resolution have yielded four different folds and as many different translocation mechanisms. The structure of the glutamate transporter homologue GltPh confirmed the role of pore-loop structures as essential parts of the translocation mechanism in one family of secondary transporters. Biochemical evidence for pore-loop structures in several other families suggest that they might be common in secondary transporters, adding to the structural and mechanistic diversity of secondary transporters.     

Peroxisomal ABC transporters

Peroxisomes perform a range of different functions, dependent upon organism, tissue type, developmental stage or environmental conditions, many of which are connected with lipid metabolism. This review summarises recent research on ATP binding cassette (ABC) transporters of the peroxisomal membrane (ABC subfamily D) and their roles in plants, fungi and animals. Analysis of mutants has revealed that peroxisomal ABC transporters play key roles in specific metabolic and developmental functions in different organisms. A common function is import of substrates for beta-oxidation but much remains to be determined concerning transport substrates and mechanisms which appear to differ significantly between phyla.     

Plant ABC transporters

The ATP binding cassette (ABC) superfamily is a large, ubiquitous and diverse group of proteins, most of which mediate transport across biological membranes. ABC transporters have been shown to function not only as ATP-dependent pumps, but also as ion channels and channel regulators. Whilst members of this gene family have been extensively characterised in mammalian and microbial systems, the study of plant ABC transporters is a relatively new field of investigation. Sequences of over 20 plant ABC proteins have been published and include homologues of P-glycoprotein, MRP, PDR5 and organellar transporters. At present, functions have been assigned to a small proportion of these genes and only the MRP subclass has been extensively characterised. This review aims to summarise literature relevant to the study of plant ABC transporters, to review methods of cloning, to discuss the utility of yeast and mammalian systems as models and to speculate on possible roles of uncharacterised ABC transporters in plants.     

Structure of ABC transporters

ABC (ATP-binding cassette) transporters are primary active membrane proteins that translocate solutes (allocrites) across lipid bilayers. The prototypical ABC transporter consists of four domains: two cytoplasmic NBDs (nucleotide-binding domains) and two TMDs (transmembrane domains). The NBDs, whose primary sequence is highly conserved throughout the superfamily, bind and hydrolyse ATP to power the transport cycle. The TMDs, whose primary sequence and protein fold can be quite disparate, form the translocation pathway across the membrane and generally (but not always) determine allocrite specificity. Structure determination of ABC proteins initially took advantage of the relative ease of expression and crystallization of the hydrophilic bacterial NBDs in isolation from the transporter complex, and revealed detailed information on the structural fold of these domains, the amino acids involved in the binding and hydrolysis of nucleotide, and the head-to-tail arrangement of the NBD-NBD dimer interface. More recently, several intact transporters have been crystallized and three types have, so far, been characterized: type I and II ABC importers, and ABC exporters. All three are present in prokaryotes, but only the ABC exporters appear to be present in eukaryotes. Their structural determination has provided insight into the mechanisms of energy and signal transduction between the NBDs and TMDs (i.e. between the ATP- and allocrite-binding sites) and, for some, the nature of the allocrite-binding site(s) within the TMDs. In this chapter, we focus primarily on the ABC exporters and describe the structural, biochemical and biophysical evidence for and against the controversial bellows-like mechanism proposed for allocrite efflux.     

Multidrug resistance ABC transporters

Clinical multidrug resistance is caused by a group of integral membrane proteins that transport hydrophobic drugs and lipids across the cell membrane. One class of these permeases, known as multidrug resistance ATP binding cassette (ABC) transporters, translocate these molecules by coupling drug/lipid efflux with energy derived from the hydrolysis of ATP. In this review, we examine both the structures and conformational changes of multidrug resistance ABC transporters. Together with the available biochemical and structural evidence, we propose a general mechanism for hydrophobic substrate transport coupled to ATP hydrolysis.     

ABC transporters in lipid transport

Since it was found that the P-glycoproteins encoded by the MDR3 (MDR2) gene in humans and the Mdr2 gene in mice are primarily phosphatidylcholine translocators, there has been increasing interest in the possibility that other ATP binding cassette (ABC) transporters are involved in lipid transport. The evidence reviewed here shows that the MDR1 P-glycoprotein and the multidrug resistance (-associated) transporter 1 (MRP1) are able to transport lipid analogues, but probably not major natural membrane lipids. Both transporters can transport a wide range of hydrophobic drugs and may see lipid analogues as just another drug. The MDR3 gene probably arose in evolution from a drug-transporting P-glycoprotein gene. Recent work has shown that the phosphatidylcholine translocator has retained significant drug transport activity and that this transport is inhibited by inhibitors of drug-transporting P-glycoproteins. Whether the phosphatidylcholine translocator also functions as a transporter of some drugs in vivo remains to be seen. Three other ABC transporters were recently shown to be involved in lipid transport: ABCR, also called Rim protein, was shown to be defective in Stargardt's macular dystrophy; this protein probably transports a complex of retinaldehyde and phosphatidylethanolamine in the retina of the eye. ABC1 was shown to be essential for the exit of cholesterol from cells and is probably a cholesterol transporter. A third example, the ABC transporter involved in the import of long-chain fatty acids into peroxisomes, is discussed in the chapter by Hettema and Tabak in this volume.     

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.     

Structure and mechanism of ABC transporters

ATP-binding cassette (ABC) transporters facilitate unidirectional translocation of chemically diverse substrates across cell or organelle membranes. The recently determined crystal structures of the vitamin B(12) importer BtuCD and its cognate binding protein BtuF have revealed critical architectural features that are probably shared by other ABC transporters. For example, the arrangement of the ABC domains and their interface with the membrane-spanning domains are probably conserved, whereas the number of transmembrane helices and their arrangement are not. Two distinct mechanistic schemes for how ABC engines couple ATP hydrolysis to substrate transport have been proposed recently and are being explored.     

The ABC transporters of Saccharomyces cerevisiae

The ABC transporters (ATP Binding Cassette) compose one of the bigest protein family with the great medical, industrial and economical impact. They are found in all organism from bacteria to man. ABC proteins are responsible for resistance of microorganism to antibiotics and fungicides and multidrug resistance of cancer cells. Mutations in ABC transporters genes cause seriuos deseases like cystic fibrosis, adrenoleucodystrophy or ataxia. Transport catalized by ABC proteins is charged with energy from the ATP hydrolysis. The ABC superfamily contains transporters, canals, receptors. Analysis of the Saccharomyces cerevisiae genome allowed to distinguish 30 potential ABC proteins which are classified into 6 subfamilies. The structural and functional similarity of the yeast and human ABC proteins allowes to use the S. cerevisiae as a model organism for ABC transporters characterisation. In this work the present state of knowleadge on yeast S. cerevisiae ABC proteins was summarised.     

Structure and mechanism of ABC transporters

ATP-binding cassette (ABC) transporters are central to many physiological processes, including the uptake of nutrients, the non-classical secretion of signaling molecules and toxins, multidrug resistance and the development of human disease. As one might expect from this spectrum of translocation events, these ubiquitous, ATP-dependent pumps or channels are capable of transporting an enormous variety of substrates, ranging from small ions to large proteins. Recently determined structures of full-length ABC transporters and isolated ABC domains have increased our understanding of the functional mechanism of these proteins.     

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.     

ABC transporters in cellular lipid trafficking

ATP-binding cassette (ABC) transporters constitute a group of evolutionary highly conserved cellular transmembrane transport proteins. Recent work has implicated ABC transporters in cellular transmembrane lipid transport and hereditary diseases have been causatively linked to defective ABC transporters translocating lipid compounds. The emerging concept that a defined subset of ABC transporters is intimately involved in cellular lipid trafficking has recently been substantiated convincingly by the finding that ABCA1 plays a central role in the regulation of HDL metabolism and macrophage targeting to the RES or the vascular wall. Differentiation dependent expression of a large number of ABC transporters in monocytes/macrophages and their regulation by sterol flux render these transporter molecules potentially critical players in atherogenesis and other chronic inflammatory diseases.     

The ATP switch model for ABC transporters

ABC transporters mediate active translocation of a diverse range of molecules across all cell membranes. They comprise two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). Recent biochemical, structural and genetic studies have led to the ATP-switch model in which ATP binding and ATP hydrolysis, respectively, induce formation and dissociation of an NBD dimer. This provides an exquisitely regulated switch that induces conformational changes in the TMDs to mediate membrane transport.     

真菌的多向耐药性ABC转运蛋白

真菌的多向耐药性ABC转运蛋白(ATP-binding cassette transporters)是导致多药耐药性和抗真菌药物治疗效果明显下降的主要原因。文章对酿酒酵母(Saccharomyces cerevisiae)和主要致病真菌白色假丝酵母(Candida albicans)、新型隐球酵母(Cryptococcus neoformans)和烟曲霉(Aspergillus fumigatus)中的多向耐药性ABC转运蛋白的种类、药物外排机制以及基因表达调控网络的研究进展作一综述,为深入了解真菌的多向耐药性机制以及探讨克服多向耐药性的策略和提高药效提供参考。     

ABC transporters and RNAi in Caenorhabditis elegans

RNAi is an evolutionarily conserved gene-silencing phenomenon that can be triggered by exogenous delivery of double stranded RNA to organisms. In Caenorhabditis elegans, the response to dsRNA is remarkably robust, and systemic RNAi responses are often observed. We have taken a genetic approach using this organism to better understand the mechanisms that facilitate RNAi. By analyzing strains of RNAi-defective mutants, we have uncovered an unexpected role for ABC transporters in RNAi and related silencing mechanisms. Ten of the sixty ABC transporter genes encoded in the C. elegans genome are required for robust RNAi. We will present data that highlights common features of these genes relative to their roles in RNAi, including genetic interactions with other components of the RNAi machinery. We will also describe unique roles for some transporter genes in endogenous RNAi-related processes.     

Evolutionary analyses of ABC transporters of Dictyostelium discoideum

The ABC superfamily of genes is one of the largest in the genomes of both bacteria and eukaryotes. The proteins encoded by these genes all carry a characteristic 200- to 250-amino-acid ATP-binding cassette that gives them their family name. In bacteria they are mostly involved in nutrient import, while in eukaryotes many are involved in export. Seven different families have been defined in eukaryotes based on sequence homology, domain topology, and function. While only 6 ABC genes in Dictyostelium discoideum have been studied in detail previously, sequences from the well-advanced Dictyostelium genome project have allowed us to recognize 68 members of this superfamily. They have been classified and compared to animal, plant, and fungal orthologs in order to gain some insight into the evolution of this superfamily. It appears that many of the genes inferred to have been present in the ancestor of the crown organisms duplicated extensively in some but not all phyla, while others were lost in one lineage or the other.     

ABC transporters in the protozoan parasite Leishmania

ATP-binding cassette (ABC) transporters constitute one of the biggest and most conserved protein families in the evolutionary scale. Many of them are of enormous clinical relevance, due to their relationship with genetic diseases and drug resistance during the treatment of cancer and infectious diseases. Leishmaniasis is a major and globally widespread group of parasitic diseases, whose treatment has been complicated by the expansion of resistance to conventional drugs. Here, we review the current knowledge about ABC transporters in Leishmania spp, with special attention to their relationship with the drug-resistance phenotype.     

ABC transporters and immunity: mechanism of self-defense

The transporter associated with antigen processing (TAP) is a prototype of an asymmetric ATP-binding cassette (ABC) transporter, which uses ATP binding and hydrolysis to translocate peptides from the cytosol to the lumen of the endoplasmic reticulum (ER). Here, we review molecular details of peptide binding and ATP binding and hydrolysis as well as the resulting allosteric cross-talk between the nucleotide-binding domains and the transmembrane domains that drive translocation of the solute across the ER membrane. We also discuss the general molecular architecture of ABC transporters and demonstrate the importance of structural and functional studies for a better understanding of the role of the noncanonical site of asymmetric ABC transporters. Several aspects of peptide binding and specificity illustrate details of peptide translocation by TAP. Furthermore, this ABC transporter forms the central part of the major histocompatibility complex class I (MHC I) peptide-loading machinery. Hence, TAP is confronted with a number of viral factors, which prevent antigen translocation and MHC I loading in virally infected cells. We review how these viral factors have been used as molecular tools to decipher mechanistic aspects of solute translocation and discuss how they can help in the structural analysis of TAP.