The YheI/YheH heterodimer from Bacillus subtilis is a multidrug ABC transporter

ABC (ATP-binding cassette) transporters form the largest family of membrane proteins in micro-organisms where they are able to transport a wide variety of substrates against a concentration gradient, in an ATP-dependent process. Two genes from the same putative Bacillus subtilis operon, yheI and yheH, encoding possibly two different ABC transporters, were overexpressed in Escherichia coli in high yield, either separately or jointly. Using membrane vesicles, it is shown here that both subunits were required to detect, (i) the transport of four structurally unrelated drugs, and (ii) a vanadate-sensitive ATPase activity. Mutation of the invariant Walker-A lysine to an alanine residue in both subunits led to an inactive transporter. Moreover, after membrane solubilization by detergent, both wild-type subunits co-purified on a Ni-Agarose affinity column while only the YheH subunit contained a hexa-histidine tag. This shows that YheI and YheH are indeed able to interact together to form a heterodimer. Importantly, expression of both yheI and yheH genes in B. subtilis could be strongly stimulated by addition of sub-inhibitory concentrations of various unrelated antibiotics. Therefore, B. subtilis YheI/YheH forms a new heterodimeric multidrug ABC transporter possibly involved in multiple antibiotic resistance in vivo.     

Complete Characterization of the Human ABC Gene Family

The human ATP-binding cassette (ABC) transporters comprise a large family of membrane transport proteins and play a vital role in many cellular processes. The genes provide functions as diverse as peptide transport, cholesterol and sterol transport, bile acid, retinoid, and iron transport. In addition some ABC genes play a role as regulatory elements. Many ABC genes play a role in human genetic diseases, and several are critical drug transport proteins overexpressed in drug resistant cells. Analysis of the gene products allows the genes to be grouped into seven different subfamilies.     

Subunit interactions in ABC transporters: towards a functional architecture

The ABC superfamily is a diverse group of integral membrane proteins involved in the ATP-dependent transport of solutes across biological membranes in both prokaryotes and eukaryotes. Although ABC transporters have been studied for over 30 years, very little is known about the mechanism by which the energy of ATP hydrolysis is used to transport substrate across the membrane. The recent report of the high resolution crystal structure of HisP, the nucleotide-binding subunit of the histidine permease complex of Salmonella typhimurium, represents a significant breakthrough toward the elucidation of the mechanism of solute translocation by ABC transporters. In this review, we use data from the crystallographic structures of HisP and other nucleotide-binding proteins, combined with sequence analysis of a subset of atypical ABC transporters, to argue a new model for the dimerisation of the nucleotide-binding domains that embraces the notion that the C motif from one subunit forms part of the ATP-binding site in the opposite subunit. We incorporate this dimerisation of the ATP-binding domains into our recently reported beta-barrel model for P-glycoprotein and present a general model for the cooperative interaction of the two nucleotide-binding domains and the translocation of mechanical energy to the transmembrane domains in ABC transporters.     

Nitensidine A,a guanidine alkaloid from Pterogyne nitens,is a novel substrate for human ABC transporter ABCB1

The Pterogyne nitens (Fabaceae) tree, native to South America, has been found to produce guanidine alkaloids as well as bioactive flavonols such as kaempferol, quercetin, and rutin. In the present study, we examined the possibility of interaction between human ATP-binding cassette (ABC) transporter ABCB1 and four guanidine alkaloids isolated from P. nitens (i.e., galegine, nitensidine A, pterogynidine, and pterogynine) using human T cell lymphoblast-like leukemia cell line CCRF-CEM and its multi-drug resistant (MDR) counterpart CEM/ADR5000. In XTT assays, CEM/ADR5000 cells were resistant to the four guanidine alkaloids compared to CCRF-CEM cells, although the four guanidine alkaloids exhibited some level of cytotoxicity against both CCRF-CEM and CEM/ADR5000 cells. In ATPase assays, three of the four guanidine alkaloids were found to stimulate the ATPase activity of ABCB1. Notably, nitensidine A was clearly found to stimulate the ATPase activity of ABCB1 as strongly as the control drug, verapamil. Furthermore, the cytotoxic effect of nitensidine A on CEM/ADR5000 cells was synergistically enhanced by verapamil. Nitensidine A inhibited the extrusion of calcein by ABCB1. In the present study, the possibility of interaction between ABCB1 and two synthetic nitensidine A analogs (nitensidine AT and AU) were examined to gain insight into the mechanism by which nitensidine A stimulates the ATPase activity of ABCB1. The ABCB1-dependent ATPase activity stimulated by nitensidine A was greatly reduced by substituting sulfur (S) or oxygen (O) for the imino nitrogen atom (N) in nitensidine A. Molecular docking studies on human ABCB1 showed that, guanidine alkaloids from P. nitens dock to the same binding pocket as verapamil. Nitensidine A and its analogs exhibit similar binding energies to verapamil. Taken together, this research clearly indicates that nitensidine A is a novel substrate for ABCB1. The present results also suggest that the number, binding site, and polymerization degree of the isoprenyl moiety in the guanidine alkaloids and the imino nitrogen atom cooperatively contribute to their stimulation of ABCB1's ATPase activity.     

ABC转运蛋白SgnA/B促进纳他霉素胞外转运与高产

纳他霉素是一种天然、广谱、高效的多烯大环内酯类还原性抗真菌剂,广泛应用于食品真菌污染的防治和临床真菌感染的治疗。纳他霉素胞外转运效率可能是限制褐黄孢链霉菌(Streptomyces gilvosporeus)发酵高产纳他霉素的重要因素。通过生物信息学及分子对接技术分析纳他霉素胞外转运蛋白SgnA/B,发现SgnA和SgnB两个异源二聚体组成的ABC转运蛋白是内向开口构象的转运蛋白,且2个结合位点与纳他霉素结合能力有强弱差异,更有利于纳他霉素的胞外转运。本研究以纳他霉素生产菌株——褐黄孢链霉菌F607为出发菌株,构建了sgnA/B基因超表达菌株F-EX,以分析sgn A/B基因超表达对纳他霉素合成及胞外转运的影响。研究发现,纳他霉素对数合成期的F-EX菌株不仅提高了纳他霉素胞外/胞内比,其120 h发酵总产量也提高了12.5%,达到7.38 g/L。最后,通过转录组测序发现,sgnA/B基因超表达除提高纳他霉素胞外转运效率外,还影响了与多种氨基酸、丙酸盐、糖、五碳化合物代谢和TCA循环相关基因的表达。研究表明,强化纳他霉素胞外转运有利于纳他霉素的合成,是提高褐黄孢链霉菌纳他霉素产量的有效...     

Disruption of AtMRP4, a guard cell plasma membrane ABCC-type ABC transporter, leads to deregulation of stomatal opening and increased drought susceptibility

ATP-binding cassette (ABC) transporters are membrane proteins responsible for cellular detoxification processes in plants and animals. Recent evidence shows that this class of transporters may also be involved in many other cellular processes. Because of their homology with human multidrug resistance-associated proteins (MRP), cystic fibrosis transmembrane conductance regulator (CFTR) and sulfonylurea receptor (SUR), some plant ABC transporters have been implicated in the regulation of ion channel activities. This paper describes an investigation of the AtMRP4 gene and its role in stomatal regulation. Reporter gene studies showed that AtMRP4 is highly expressed in stomata and that the protein is localized to the plasma membrane. Stomatal aperture in three independent atmrp4 mutant alleles was larger than in wild-type plants, both in the light and in the dark, resulting in increased water loss but no change in the photosynthetic rate. In baker's yeast, AtMRP4 shows ATP-dependent, vanadate-sensitive transport of methotrexate (MTX), an antifolate and a substrate of mammalian MRPs. Treatment with MTX reduced stomatal opening in wild-type plants, but had no effect in atmrp4 mutants. These results indicate the involvement of AtMRP4 in the complex regulation of stomatal aperture.     

Protein Linkers Provide Limits on the Domain Interactions in the ABC Importer GlnPQ and Determine the Rate of Transport

GlnPQ is an ATP-binding cassette importer with a unique domain organization and intricate transport behavior. The protein has two extracytoplamic substrate-binding domains (SBDs) per membrane subunit, each with different specificity for amino acids and different spacing to the translocator domain. We determined the effect of the length and structure of the linkers, which connect the SBDs to each other and to the membrane-embedded translocator domain, on the transport by GlnPQ. We reveal that varying the linker length impacts transport in a dual manner that depends on the conformational dynamics of the SBD. Varying the linker length not only changes the time for the SBD to find the translocator (docking) but also changes the probability to release the substrate again, thus altering the transport efficiency. On the basis of the experimental data and mathematical modeling, we calculate the docking efficiency as function of linker length and lifetime of the closed conformation. Importantly, not only linker length but also features in the sequence are important for efficient delivery of substrate from SBD to the translocator. We show that the linkers provide a platform for SBD docking and are not merely flexible structures.     

ABC transporter architecture and mechanism: implications from the crystal structures of BtuCD and BtuF

ABC transporters are ubiquitous membrane proteins that facilitate unidirectional substrate translocation across the lipid bilayer. Over the past five years, new crystal structures have advanced our understanding of how ABC transporters couple adenosine triphosphate (ATP) hydrolysis to substrate transport. In the following, we will briefly review the results of these structural investigations and outline their mechanistic implications.     

Characterization of ABC transporter ABCB1 expressed in human neural stem/progenitor cells

We investigated the localization and functional expression of the ABC transporter ABCB1 in human fetal neural stem/progenitor cells (hNSPCs). RT-PCR analysis revealed ABCB1 gene expression in hNSPCs. We found a single band in immunoblotted hNSPCs lysates probed with ABCB1 antibody, and detected ABCB1 at the hNSPCs cell membrane by immunocytochemistry and subcellular fractionation. ABCB1 inhibitors and substrate, and ATP-depleting agents enhanced hNSPCs' rhodamine 123 accumulation, and hNSPCs microsomes had vanadate-sensitive ATPase activity. ABCB1 and nestin expression decreased during hNSPCs differentiation, while the astroglial marker GFAP increased. ABCB1 may maintain hNSPCs in an undifferentiated state and could be a neural stem/progenitor marker.     

Mitochondrial ABC proteins in health and disease

ABC transporters represent one of the largest families of membrane proteins that are found in all three phyla of life. Mitochondria comprise up to four ABC systems, ABCB7/ATM1, ABCB10/MDL1, ABCB8 and ABCB6. These half-transporters, which assemble into homodimeric complexes, are involved in a number of key cellular processes, e.g. biogenesis of cytosolic iron-sulfur clusters, heme biosynthesis, iron homeostasis, multidrug resistance, and protection against oxidative stress. Here, we summarize recent advances and emerging themes in our understanding of how these ABC systems in the inner and outer mitochondrial membrane fulfill their functions in important (patho) physiological processes, including neurodegenerative and hematological disorders.     

Thermophilic Adaptation of Proteins

Referee: Dr. Ruth Nussinov, Saic Frederick, Bldg. 469. 469, Room 151, Frederick, MD 21702-1201

Hyperthermophilic organisms optimally grow close to the boiling point of water. As a consequence, their macromolecules must be much more thermostable than those from mesophilic species. Here, proteins from hyperthermophiles and mesophiles are compared with respect to their thermodynamic and kinetic stabilities. The known differences in amino acid sequences and three-dimensional structures between intrinsically thermostable and thermolabile proteins will be summarized, and the crucial role of electrostatic interactions for protein stability at high temperatures will be highlighted. Successful attempts to increase the thermostability of proteins, which were either based on rational design or on directed evolution, are presented. The relationship between high thermo-stability of enzymes from hyperthermophiles and their low catalytic activity at room temperature is discussed. Not all proteins from hyperthermophiles are thermostable enough to retain their structures and functions at the high physiological temperatures. It will be shown how this shortcoming can be surpassed by extrinsic factors such as large molecular chaperones and small compatible solutes. Finally, the potential of thermostable enzymes for biotechnology is discussed.     

Genome-wide expression analysis of rice ABC transporter family across spatio-temporal samples and in response to abiotic stresses

Although the super family of ATP-binding cassette (ABC) proteins plays key roles in the physiology and development of plants, the functions of members of this interesting family mostly remain to be clarified, especially in crop plants. Thus, systematic analysis of this family in rice (Oryza sativa), a major model crop plant, will be helpful in the design of effective strategies for functional analysis. Phylogenomic analysis that integrates anatomy and stress meta-profiling data based on a large collection of rice Affymetrix array data into the phylogenic context provides useful clues into the functions for each of the ABC transporter family members in rice. Using anatomy data, we identified 17 root-preferred and 16-shoot preferred genes at the vegetative stage, and 3 pollen, 2 embryo, 2 ovary, 2 endosperm, and 1 anther-preferred gene at the reproductive stage. The stress data revealed significant up-regulation or down-regulation of 47 genes under heavy metal treatment, 16 genes under nutrient deficient conditions, and 51 genes under abiotic stress conditions. Of these, we confirmed the differential expression patterns of 14 genes in root samples exposed to drought stress using quantitative real-time PCR. Network analysis using RiceNet suggests a functional gene network involving nine rice ABC transporters that are differentially regulated by drought stress in root, further enhancing the prediction of biological function. Our analysis provides a molecular basis for the study of diverse biological phenomena mediated by the ABC family in rice and will contribute to the enhancement of crop yield and stress tolerance.     

Lipid dependence of ABC transporter localization and function

Lipid rafts have been implicated in many cellular functions, including protein and lipid transport and signal transduction. ATP-binding cassette (ABC) transporters have also been localized in these membrane domains. In this review the evidence for this specific localization will be evaluated and discussed in terms of relevance to ABC transporter function. We will focus on three ABC transporters of the A, B and C subfamily, respectively. Two of these transporters are relevant to multidrug resistance in tumor cells (Pgp/ABCB1 and MRP1/ABCC1), while the third (ABCA1) is extensively studied in relation to the reverse cholesterol pathway and cellular cholesterol homeostasis. We will attempt to derive a generalized model of lipid rafts to which they associate based on the use of various different lipid raft isolation procedures. In the context of lipid rafts, modulation of ABC transporter localization and function by two relevant lipid classes, i.e. sphingolipids and cholesterol, will be discussed.     

ABC multidrug transporter Cdr1p of Candida albicans has divergent nucleotide-binding domains which display functional asymmetry

In order to ascertain the molecular basis of ATP-mediated drug extrusion by Cdr1p, a multidrug transporter of Candida albicans, we recently have reported that the Walker A motif of the N-terminal nucleotide biding domain (NBD) of this protein contains an uncommon cysteine residue (C193; GXXGXGCS/T) which is indispensable for ATP hydrolysis. This residue is exceptionally conserved in N-terminal NBDs of fungal ABC transporters and hence makes these transporters an evolutionarily divergent group. However, the presence of a conventional lysine residue at a similar position in the Walker A motif of the C-terminal NBD warrants the individual contribution of both the NBDs in the ATP-driven efflux function of such transporters. In this study we have investigated the contribution of this divergent Walker A motif in the context of the full Cdr1p protein under in vivo conditions by swapping these two crucial amino acids (C193K in Walker A motif of N-terminal NBD and K901C in Walker A motif of C-terminal NBD) between the two NBDs. Both the native and the mutant variants of Cdr1p were integrated at the PDR5 locus as GFP-tagged fusion proteins and were hyper-expressed. Our study shows that both C193K- and K901C-expressing cells elicit a severe impairment of Cdr1p's ATPase function. However, both these mutations have distinct phenotypes with respect to other functional parameters such as substrate efflux and drug resistance profiles. In contrast to C193K, K901C mutant cells were substantially hypersensitive to the tested drugs (fluconazole, ansiomycin, miconazole and cycloheximide) and were unable to expel rhodamine 6G. Our results for the first time show that both NBDs influence the Cdr1p function asymmetrically, and that the positioning of the cysteine and lysine residues within the respective Walker A motifs is functionally not interchangeable.     

ABC transporters as cancer drivers: Potential functions in cancer development

Background

ABC transporters have attracted considerable attention for their function as drug transporters in a broad range of tumours and are therefore considered as major players in cancer chemoresistance. However, less attention has been focused on their potential role as active players in cancer development and progression.

Dynamics of ATP-binding cassette contribute to allosteric control, nucleotide binding and energy transduction in ABC transporters

ATP-binding cassette (ABC) transporters move solutes across membranes and are associated with important diseases, including cystic fibrosis and multi-drug resistance. These molecular machines are energized by their charateristic ABC modules, molecular engines fuelled by ATP hydrolysis. A solution NMR study of a model ABC, Methanococcus jannaschii protein MJ1267, reveals that ADP-Mg binding alters the flexibilities of key ABC motifs and induces allosteric changes in conformational dynamics in the LivG insert, over 30A away from the ATPase active site. (15)N spin relaxation data support a "selected-fit" model for nucleotide binding. Transitions between rigidity and flexibility in key motifs during the ATP hydrolysis cycle may be crucial to mechanochemical energy transduction in ABC transporters. The restriction of correlated protein motions is likely a central mechanism for allosteric communications. Comparison between dynamics data from NMR and X-ray crystallography reveals their overall consistency and complementarity.     

Functional reconstitution and osmoregulatory properties of the ProU ABC transporter from Escherichia coli

Abstract

The ATP-binding cassette (ABC) transporter ProU from Escherichia coli translocates a wide range of compatible solutes and contributes to the regulation of cell volume, which is particularly important when the osmolality of the environment fluctuates. We have purified the components of ProU, i.e., the substrate-binding protein ProX, the nucleotide-binding protein ProV and the transmembrane protein ProW, and reconstituted the full transporter complex in liposomes. We engineered a lipid anchor to ProX for surface tethering of this protein to ProVW-containing proteoliposomes. We show that glycine betaine binds to ProX with high-affinity and is transported via ProXVW in an ATP-dependent manner. The activity ProU is salt and anionic lipid-dependent and mimics the ionic strength-gating of transport of the homologous OpuA system.