The yeast Pdr15p ATP-binding cassette (ABC) protein is a general stress response factor implicated in cellular detoxification

ATP-binding cassette (ABC) transporters play important roles in drug efflux, but some may also function in cellular detoxification. The Pdr15p ABC protein is the closest homologue of the multidrug efflux transporter Pdr5p, which mediates pleiotropic drug resistance to hundreds of unrelated compounds. In this study, we show that the plasma membrane protein Pdr15p displays limited drug transport capacity, mediating chloramphenicol and detergent tolerance. Interestingly, Pdr15p becomes most abundant when cells exit the exponential growth phase, whereas its closest homologue, Pdr5p, disappears after exponential growth. Furthermore, in contrast to Pdr5p, Pdr15p is strongly induced by various stress conditions including heat shock, low pH, weak acids, or high osmolarity. PDR15 induction bypasses the Pdr1p/Pdr3p regulators but requires the general stress regulator Msn2p, which directly decorates the stress response elements in the PDR15 promoter. Remarkably, however, Pdr15p induction bypasses upstream components of the high osmolarity glycerol (HOG) pathway including the Hog1p and Pbs2p kinases as well as the dedicated HOG cell surface sensors. Our data provide evidence for a novel upstream branch of the general stress response pathway activating Msn2p. In addition, the results demonstrate a cross-talk between stress response and the pleiotropic drug resistance network.     

The human ATP-binding cassette (ABC) transporter superfamily.

The transport of specific molecules across lipid membranes is an essential function of all living organisms and a large number of specific transporters have evolved to carry out this function. The largest transporter gene family is the ATP-binding cassette (ABC) transporter superfamily. These proteins translocate a wide variety of substrates including sugars, amino acids, metal ions, peptides, and proteins, and a large number of hydrophobic compounds and metabolites across extra- and intracellular membranes. ABC genes are essential for many processes in the cell, and mutations in these genes cause or contribute to several human genetic disorders including cystic fibrosis, neurological disease, retinal degeneration, cholesterol and bile transport defects, anemia, and drug response. Characterization of eukaryotic genomes has allowed the complete identification of all the ABC genes in the yeast Saccharomyces cerevisiae, Drosophila, and C. elegans genomes. To date, there are 48 characterized human ABC genes. The genes can be divided into seven distinct subfamilies, based on organization of domains and amino acid homology. Many ABC genes play a role in the maintenance of the lipid bilayer and in the transport of fatty acids and sterols within the body. Here, we review the current knowledge of the human ABC genes, their role in inherited disease, and understanding of the topology of these genes within the membrane.     

Expression of the ATP-binding cassette transporter gene ABCG1 (ABC8) in Tangier disease

Several members of the ATP-binding cassette (ABC) transporter family are involved in cholesterol efflux from cells. A defect in one member, ABCA1, results in Tangier disease, a condition characterized by cholesterol accumulation in macrophages and virtual absence of mature circulating high-density lipoproteins. Expression of a second member, ABCG1, is increased by cholesterol-loading in human macrophages. We now show that ABCG1, which we identified by differential display RT-PCR in foamy macrophages, is overexpressed in macrophages from patients with Tangier disease compared to control macrophages. On examination by confocal laser scanning microscopy, ABCG1 was present in perinuclear structures within the cell. In addition, a combination of in situ hybridization and indirect immunofluorescence microscopy revealed that ABCG1 is expressed in foamy macrophages within the atherosclerotic plaque. These data indicate that not only ABCA1 but also ABCG1 may play a role in the cholesterol metabolism of macrophages in vitro and in the atherosclerotic plaque.     

Unravelling the folding and stability of an ABC (ATP-binding cassette) transporter

Prokaryotic importers from the large family of ABC (ATP-binding cassette) transporters comprise four separate subunits: two membrane-embedded and two cytoplasmic ATP-binding subunits. This modular construction makes them ideal candidates for studies of the intersubunit interactions of membrane protein complexes that contain both hydrophobic and hydrophilic subunits. In the present paper, we focus on the vitamin B12 importer of Escherichia coli, BtuCD, that contains two transmembrane BtuC subunits and two ATP-binding BtuD subunits. We have studied the factors that induce subunit dissociation and unfolding in vitro. The BtuCD complex remains intact in alcohol and mild detergents, but urea or SDS separate the BtuC and BtuD subunits, with 6?M urea causing 80% of BtuD to be removed from BtuCD. ATP is found to stabilize the complex as a result of its binding to the BtuD subunits. In the absence of ATP, low concentrations of urea (0.5-3?M) also induce some unfolding, with approximately 14% reduction in helicity in 3?M urea, whereas, in the presence of ATP, no changes are observed. Disassembly at the BtuD-BtuD dimeric interface in BtuCD can be achieved with smaller concentrations of urea (0.5-3?M) than that required to cause disassembly at the BtuC-BtuD transmission interface (3-8?M), suggesting a stronger interaction of the latter. The results also suggest that unfolding and disassociation of subunits appear to be coupled processes. Our work provides insights into the subunit interactions of an ABC transporter and lays the foundation for studies of the reassembly of BtuCD.     

The ATP/substrate stoichiometry of the ATP-binding cassette (ABC) transporter OpuA

ATP-binding cassette (ABC) transport proteins catalyze the translocation of substrates at the expense of hydrolysis of ATP, but the actual ATP/substrate stoichiometry is still controversial. In the osmoregulated ABC transporter (OpuA) from Lactococcus lactis, ATP hydrolysis and substrate translocation are tightly coupled, and the activity of right-side-in and inside-out reconstituted OpuA can be determined accurately. Although the ATP/substrate stoichiometry determined from the uptake of glycine betaine and intravesicular ATP hydrolysis tends to increase with decreasing average size of the liposomes, the data from inside-out reconstituted OpuA indicate that the mechanistic stoichiometry is 2. Moreover, the two orientations of OpuA in proteoliposomes allowed possible contributions from substrate (glycine betaine) inhibition on the trans-side of the membrane and inhibition by ADP to be determined. Here we show that OpuA is not inhibited by up to 400 mm glycine betaine on the trans-side of the membrane. ADP is an inhibitor, but accumulation of ADP was negligible in the assays with inside-out-oriented OpuA, and potential effects of the ATP/ADP ratio on the ATP/substrate stoichiometry determinations could be eliminated.     

Uptake of the ATP-binding cassette (ABC) transporter Ste6 into the yeast vacuole is blocked in the doa4 Mutant

Previous experiments suggested that trafficking of the a-factor transporter Ste6 of Saccharomyces cerevisiae to the yeast vacuole is regulated by ubiquitination. To define the ubiquitination-dependent step in the trafficking pathway, we examined the intracellular localization of Ste6 in the ubiquitination-deficient doa4 mutant by immunofluorescence experiments, with a Ste6-green fluorescent protein fusion protein and by sucrose density gradient fractionation. We found that Ste6 accumulated at the vacuolar membrane in the doa4 mutant and not at the cell surface. Experiments with a doa4 pep4 double mutant showed that Ste6 uptake into the lumen of the vacuole is inhibited in the doa4 mutant. The uptake defect could be suppressed by expression of additional ubiquitin, indicating that it is primarily the result of a lowered ubiquitin level (and thus of reduced ubiquitination) and not the result of a deubiquitination defect. Based on our findings, we propose that ubiquitination of Ste6 or of a trafficking factor is required for Ste6 sorting into the multivesicular bodies pathway. In addition, we obtained evidence suggesting that Ste6 recycles between an internal compartment and the plasma membrane.     

Genome-wide analysis of the ATP-binding cassette (ABC) transporter gene family in the silkworm, Bombyx mori

The ATP-binding cassette (ABC) superfamily is a larger protein family with diverse physiological functions in all kingdoms of life. We identified 53 ABC transporters in the silkworm genome, and classified them into eight subfamilies (A-H). Comparative genome analysis revealed that the silkworm has an expanded ABCC subfamily with more members than Drosophila melanogaster, Caenorhabditis elegans, or Homo sapiens. Phylogenetic analysis showed that the ABCE and ABCF genes were highly conserved in the silkworm, indicating possible involvement in fundamental biological processes. Five multidrug resistance-related genes in the ABCB subfamily and two multidrug resistance-associated-related genes in the ABCC subfamily indicated involvement in biochemical defense. Genetic variation analysis revealed four ABC genes that might be evolving under positive selection. Moreover, the silkworm ABCC4 gene might be important for silkworm domestication. Microarray analysis showed that the silkworm ABC genes had distinct expression patterns in different tissues on day 3 of the fifth instar. These results might provide new insights for further functional studies on the ABC genes in the silkworm genome.     

Mapping putative contact sites between subunits in a bacterial ATP-binding cassette (ABC) transporter by synthetic peptide libraries

The maltose ATP-binding cassette transporter of Salmonella typhimurium is composed of the soluble periplasmic receptor, MalE, and a membrane-associated complex comprising one copy each of the pore-forming hydrophobic subunits, MalF and MalG, and of a homodimer of the ATP-hydrolyzing subunit, MalK. During the transport process the subunits are thought to undergo conformational changes that might transiently alter molecular contacts between MalFG and MalK(2). In order to map sites of subunit-subunit interactions we have used a comprehensive peptide mapping approach comprising large-scale microsynthesis of labelled probes and array techniques. In particular, we screened the binding of (i) MalFG-derived soluble biotinylated peptides to immobilized MalK, and (ii) radiolabelled MalK to MalFG-derived cellulose membrane-bound peptides. The first approach identified seven peptides (10mers) each of MalF and MalG that specifically bound to MalK. The peptides were localized to TMDs 3 and 6, periplasmic loop P4 and cytoplasmic loops C2 and C3 of MalF, while MalG-derived peptides localized to the N terminus, TMDs 4-6, periplasmic loop P1 and cytoplasmic loop C2. Peptides from C3 and C2, respectively, of MalF and MalG partially encompass the conserved EAA-motif, known to be crucial for interaction with MalK. These results were basically confirmed by screening MalFG-derived peptide arrays consisting of 16mers or 31mers with radiolabelled MalK. This approach also allowed us to perform complete substitutional analyses of peptides in question. The results led to the construction of MalFG variants that were subsequently analyzed for functional consequences in vivo. Growth experiments revealed that most of the mutations had no phenotype, suggesting that the mutated residues themselves are not critical but part of a discontinuous binding site. However, two novel mutations affecting residues from the EAA motifs of MalF (Ile417Glu) and MalG (Phe203Gln/Asn), respectively, displayed severe growth defects, indicating their functional importance. Together, these experimental outcomes identify specific molecular contacts made between MalK and MalFG that extend beyond the well-characterized EAA motif.     

ATP-binding cassette (ABC) transporters in human metabolism and diseases

The ATP-binding cassette (ABC) superfamily of active transporters involves a large number of functionally diverse transmembrane proteins. They transport a variety of substrates including amino acids, lipids, inorganic ions, peptides, saccharides, metals, drugs, and proteins. The ABC transporters not only move a variety of substrates into and out of the cell, but also are also involved in intracellular compartmental transport. Energy derived from the hydrolysis of ATP is used to transport the substrate across the membrane against a concentration gradient. The typical ABC transporter consists of two transmembrane domains and two nucleotide-binding domains. Defects in 14 of these transporters cause 13 genetic diseases (cystic fibrosis, Stargardt disease, adrenoleukodystrophy, Tangier disease, etc.). Mutations in three genes affect lipid levels expressively. Mutations in ABCA1 cause severe HDL deficiency syndromes called Tangier disease and familial high-density lipoprotein deficiency, which are characterized by a severe deficiency or absence of high-density lipoprotein in the plasma. Two other ABCG transporters, ABCG5 and ABCG8, mutations of which cause sitosterolemia, have been identified. The affected individuals absorb and retain plant sterols, as well as shellfish sterols.     

ATP-binding cassette (ABC) transporters in normal and pathological lung

ATP-binding cassette (ABC) transporters are a family of transmembrane proteins that can transport a wide variety of substrates across biological membranes in an energy-dependent manner. Many ABC transporters such as P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1) and breast cancer resistance protein (BCRP) are highly expressed in bronchial epithelium. This review aims to give new insights in the possible functions of ABC molecules in the lung in view of their expression in different cell types. Furthermore, their role in protection against noxious compounds, e.g. air pollutants and cigarette smoke components, will be discussed as well as the (mal)function in normal and pathological lung. Several pulmonary drugs are substrates for ABC transporters and therefore, the delivery of these drugs to the site of action may be highly dependent on the presence and activity of many ABC transporters in several cell types. Three ABC transporters are known to play an important role in lung functioning. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene can cause cystic fibrosis, and mutations in ABCA1 and ABCA3 are responsible for respectively Tangier disease and fatal surfactant deficiency. The role of altered function of ABC transporters in highly prevalent pulmonary diseases such as asthma or chronic obstructive pulmonary disease (COPD) have hardly been investigated so far. We especially focused on polymorphisms, knock-out mice models and in vitro results of pulmonary research. Insight in the function of ABC transporters in the lung may open new ways to facilitate treatment of lung diseases.     

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.     

Structure of the ATPase subunit CysA of the putative sulfate ATP-binding cassette (ABC) transporter from Alicyclobacillus acidocaldarius

CysA, the ATPase subunit of a putative sulfate ATP-binding cassette transport system of the gram-positive thermoacidophilic bacterium Alicyclobacillus acidocaldarius, was structurally characterized at a resolution of 2.0 Angstroms in the absence of nucleotides. In line with previous findings on ABC-ATPases the structures of the two monomers (called CysA-1 and CysA-2) in the asymmetric unit differ substantially in the arrangement of their individual (sub)domains. CysA-2 was found as a physiological dimer composed of two crystallographically related monomers that are arranged in an open state. Interestingly, while the regulatory domain of CysA-2 packs against its opposing domain that of CysA-1 undergoes a conformational change and, in the dimer, would interfere with the opposing monomer thereby preventing solute translocation. Whether this conformational state is used for regulatory purposes will be discussed.     

Isolation and characterization of the ATP-binding cassette (ABC) transporter system genes from loofah witches' broom phytoplasma.

A clone containing a 3903 bp EcoRI-restriction fragment was obtained from a lambda(ZAP) genomic library of loofah witches' broom (LfWB) phytoplasma by plaque hybridization using a PCR fragment as a probe. Sequence analysis revealed that this fragment contained three open reading frames (ORFs). The deduced amino acid sequences of ORF 1 and ORF 2 showed a high homology with the ATP-binding proteins of the ABC transporter system genes of prokaryotes and eukaryotes, and encoded proteins with a molecular mass of 36 and 30 kDa, respectively. Based on amino acid sequence similarity, secondary structure, hydrophilicity and a signal peptide sequence at the N-terminus, we predicted that ORF 3 might encode a specific solute-binding prolipoprotein of the ABC transporter system with a molecular mass of 62 kDa. The cleavage site of this prolipoprotein signal peptide was similar to those of gram-positive bacteria. In addition to nutrient uptake, ABC transporter systems of bacteria also play a role in signal transduction, drug-resistance and perhaps virulence. The possible implications of the system to the survival and the pathogenesis of phytoplasma were discussed.     

Role of the yeast ABC transporter Yor1p in cadmium detoxification

Growth of yeast strains, either deleted for the vacuolar ABC transporter Ycf1 or deleted for the plasma membrane ABC transporter Yor1p or overexpressing Yor1p, were compared for their sensitivity to cadmium. On solid medium cell death (or growth inhibition) was observed at cadmium concentrations higher than 100 microM when yeasts were grown at 30 degrees C for 24 h. However, for all tested strains cell death (or growth inhibition) was already observed at 40 microM cadmium when incubated at 23 degrees C for 60 h. Thus cadmium is more toxic to yeast at 23 degrees C than at 30 degrees C. At 23 degrees C, the Deltayor1 strain grew more slowly than the wild-type strain and the double Deltayor1, Deltaycf1 deleted strain was much more sensitive to cadmium than each single Deltayor1 or Deltaycf1 deletant. Overexpression of Yor1p in a Deltaycf1 strain restores full growth. Cadmium uptake measurements show that Deltaycf1 yeast strains expressing or overexpressing Yor1p store less cadmium than the corresponding Deltaycf1, Deltayor1 strain. The strains expressing Yor1p display an energy-dependent efflux of cadmium estimated for the yeast overexpressing Yor1p to be about 0.02 nmol 109Cd/mg protein/min. Yeast cells loaded with radiolabeled glutathione and then with radioactive cadmium displayed a twice-higher efflux of glutathione than that of cadmium suggesting that Yor1p transports both compounds as a bis-glutathionato-cadmium complex. All together, these results suggest that in addition to being accumulated in the yeast vacuole by Ycf1p, cadmium is also effluxed out of the cell by Yor1p.     

Identification of a novel human sterol-sensitive ATP-binding cassette transporter (ABCA7)

We report the identification of the full-length cDNA for a novel ATP-binding cassette (ABC) transporter from human macrophages. The mRNA is of 6.8 kb size and contains an open reading frame encoding a polypeptide of 2146 amino acids with a calculated molecular weight of 220 kDa. The predicted protein product is composed of two transmembrane domains and two nucleotide binding folds indicating that it pertains to the group of full-size ABC transporters. The novel transporter shows highest protein sequence homology with the recently cloned human cholesterol and phospholipid exporter ABCA1 (54%) and the human retinal transporter ABCR (49%), both members of the ABC transporter subfamily A. In accordance with the currently proposed classification, the novel transporter was designated ABCA7. ABCA7 mRNA was detected predominantly in myelo-lymphatic tissues with highest expression in peripheral leukocytes, thymus, spleen, and bone marrow. Expression of ABCA7 is induced during in vitro differentiation of human monocytes into macrophages. In macrophages, both the ABCA7 mRNA and protein expression are upregulated in the presence of modified low density lipoprotein and downregulated by HDL(3). Our results suggest a role for ABCA7 in macrophage transmembrane lipid transport.     

Transmembrane gate movements in the type II ATP-binding cassette (ABC) importer BtuCD-F during nucleotide cycle

ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins that translocate substrates across cell membranes. The alternating access of their transmembrane domains to opposite sides of the membrane powered by the closure and reopening of the nucleotide binding domains is proposed to drive the translocation events. Despite clear structural similarities, evidence for considerable mechanistic diversity starts to accumulate within the importers subfamily. We present here a detailed study of the gating mechanism of a type II ABC importer, the BtuCD-F vitamin B(12) importer from Escherichia coli, elucidated by EPR spectroscopy. Distance changes at key positions in the translocation gates in the nucleotide-free, ATP- and ADP-bound conformations of the transporter were measured in detergent micelles and liposomes. The translocation gates of the BtuCD-F complex undergo conformational changes in line with a "two-state" alternating access model. We provide the first direct evidence that binding of ATP drives the gates to an inward-facing conformation, in contrast to type I importers specific for maltose, molybdate, or methionine. Following ATP hydrolysis, the translocation gates restore to an apo-like conformation. In the presence of ATP, an excess of vitamin B(12) promotes the reopening of the gates toward the periplasm and the dislodgment of BtuF from the transporter. The EPR data allow a productive translocation cycle of the vitamin B(12) transporter to be modeled.     

Nucleotide-dependent allostery within the ABC transporter ATP-binding cassette: a computational study of the MJ0796 dimer

ATP-binding cassette transporters perform energy-dependent transmembrane solute trafficking in all organisms. These proteins often mediate cellular resistance to therapeutic drugs and are involved in a range of human genetic diseases. Enzymological studies have implicated a helical subdomain within the ATP-binding cassette nucleotide-binding domain in coupling ATP hydrolysis to solute transport in the transmembrane domains. Consistent with this, structural and computational analyses have indicated that the helical subdomain undergoes nucleotide-dependent movement relative to the core of the nucleotide-binding domain fold. Here we use theoretical methods to examine the allosteric nucleotide dependence of helical subdomain transitions to further elucidate its role in interactions between the transmembrane and nucleotide-binding domains. Unrestrained 30-ns molecular dynamics simulations of the ATP-bound, ADP-bound, and apo states of the MJ0796 monomer support the idea that interaction of a conserved glutamine residue with the catalytic metal mediates the rotation of the helical subdomain in response to nucleotide binding and hydrolysis. Simulations of the nucleotide-binding domain dimer revealed that ATP hydrolysis induces a large transition of one helical subdomain, resulting in an asymmetric conformation of the dimer not observed previously. A coarse-grained elastic network analysis supports this finding, revealing the existence of corresponding dynamic modes intrinsic to the contact topology of the protein. The implications of these findings for the coupling of ATP hydrolysis to conformational changes in the transmembrane domains required for solute transport are discussed in light of recent whole transporter structures.     

Conformational transitions induced by the binding of MgATP to the vitamin B12 ATP-binding cassette (ABC) transporter BtuCD

ATP-binding cassette transporters use the free energy of ATP hydrolysis to transport structurally diverse molecules across prokaryotic and eukaryotic membranes. Computer simulation studies of the "real-time" dynamics of the ATP binding process in BtuCD, the vitamin B12 importer from Escherichia coli, demonstrate that the docking of ATP to the catalytic pockets progressively draws the two cytoplasmic nucleotide-binding cassettes toward each other. Movement of the cassettes into closer opposition in turn induces conformational rearrangement of alpha-helices in the transmembrane domain. The shape of the translocation pathway consequently changes in a manner that could aid the vectorial movement of vitamin B12. These results suggest that ATP binding may indeed represent the power stroke in the catalytic mechanism. Moreover, occlusion of ATP at one catalytic site is mechanically coupled to opening of the nucleotide-binding pocket at the second site. We propose that this asymmetry in nucleotide binding behavior at the two catalytic pockets may form the structural basis by which the transporter is able to alternate ATP hydrolysis from one site to the other.