Whole-Genome Survey of the Putative ATP-Binding Cassette Transporter Family Genes in Vitis vinifera

The ATP-binding cassette (ABC) protein superfamily constitutes one of the largest protein families known in plants. In this report, we performed a complete inventory of ABC protein genes in Vitis vinifera, the whole genome of which has been sequenced. By comparison with ABC protein members of Arabidopsis thaliana, we identified 135 putative ABC proteins with 1 or 2 NBDs in V. vinifera. Of these, 120 encode intrinsic membrane proteins, and 15 encode proteins missing TMDs. V. vinifera ABC proteins can be divided into 13 subfamilies with 79 “full-size,” 41 “half-size,” and 15 “soluble” putative ABC proteins. The main feature of the Vitis ABC superfamily is the presence of 2 large subfamilies, ABCG (pleiotropic drug resistance and white-brown complex homolog) and ABCC (multidrug resistance-associated protein). We identified orthologs of V. vinifera putative ABC transporters in different species. This work represents the first complete inventory of ABC transporters in V. vinifera. The identification of Vitis ABC transporters and their comparative analysis with the Arabidopsis counterparts revealed a strong conservation between the 2 species. This inventory could help elucidate the biological and physiological functions of these transporters in V. vinifera.     

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.     

Asymmetric Conformational Flexibility in the ATP-Binding Cassette Transporter HI1470/1

Putative metal-chelate-type ABC transporter HI1470/1 is homologous with vitamin B₁₂ importer BtuCD but exhibits a distinct inward-facing conformation in contrast to the outward-facing conformation of BtuCD. Normal-mode analysis of HI1470/1 reveals the intrinsic asymmetric conformational flexibility in this transporter and demonstrates that the transition from the inward-facing to the outward-facing conformation is realized through the asymmetric motion of individual subunits of the transporter. This analysis suggests that the asymmetric arrangement of the BtuC dimer in the crystal structure of the BtuCD-F complex represents an intermediate state relating HI1470/1 and BtuCD. Furthermore, a twisting motion between transmembrane domains and nucleotide-binding domains encoded in the lowest-frequency normal mode of this type of importer is found to contribute to the conformational transitions during the whole cycle of substrate transportation. A more complete translocation mechanism of the BtuCD type importer is proposed.     

Generating Symmetry in the Asymmetric ATP-binding Cassette (ABC) Transporter Pdr5 from Saccharomyces cerevisiae

Pdr5 is a plasma membrane-bound ABC transporter from Saccharomyces cerevisiae and is involved in the phenomenon of resistance against xenobiotics, which are clinically relevant in bacteria, fungi, and humans. Many fungal ABC transporters such as Pdr5 display an inherent asymmetry in their nucleotide-binding sites (NBS) unlike most of their human counterparts. This degeneracy of the NBSs is very intriguing and needs explanation in terms of structural and functional relevance. In this study, we mutated nonconsensus amino acid residues in the NBSs to its consensus counterpart and studied its effect on the function of the protein and effect on yeast cells. The completely “regenerated” Pdr5 protein was severely impaired in its function of ATP hydrolysis and of rhodamine 6G transport. Moreover, we observe alternative compensatory mechanisms to counteract drug toxicity in some of the mutants. In essence, we describe here the first attempts to restore complete symmetry in an asymmetric ABC transporter and to study its effects, which might be relevant to the entire class of asymmetric ABC transporters.     

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     

The Allosteric Regulatory Mechanism of the Escherichia coli MetNI Methionine ATP Binding Cassette (ABC) Transporter

The MetNI methionine importer of Escherichia coli, an ATP binding cassette (ABC) transporter, uses the energy of ATP binding and hydrolysis to catalyze the high affinity uptake of d- and l-methionine. Early in vivo studies showed that the uptake of external methionine is repressed by the level of the internal methionine pool, a phenomenon termed transinhibition. Our understanding of the MetNI mechanism has thus far been limited to a series of crystal structures in an inward-facing conformation. To understand the molecular mechanism of transinhibition, we studied the kinetics of ATP hydrolysis using detergent-solubilized MetNI. We find that transinhibition is due to noncompetitive inhibition by l-methionine, much like a negative feedback loop. Thermodynamic analyses revealed two allosteric methionine binding sites per transporter. This quantitative analysis of transinhibition, the first to our knowledge for a structurally defined transporter, builds upon the previously proposed structurally based model for regulation. This mechanism of regulation at the transporter activity level could be applicable to not only ABC transporters but other types of membrane transporters as well.     

Molecular Characterization of LjABCG1, an ATP-Binding Cassette Protein in Lotus japonicus

LjABCG1, a full-size ABCG subfamily of ATP-binding cassette proteins of a model legume, Lotus japonicus, was reported as a gene highly expressed during the early stages of nodulation, but have not been characterized in detail. In this study we showed that the induction of LjABCG1 expression was remarkable by methyl jasmonate treatment, and reporter gene experiments indicated that LjABCG1 was strongly expressed in the nodule parenchyma and cell layers adjacent to the root vascular tissue toward the nodule. LjABCG1 was suggested to be localized at the plasma membrane based on the fractionation of microsomal membranes as well as separation via aqueous two-phase partitioning. The physiological functions of LjABCG1 in symbiosis and pathogenesis were analyzed in homologous and heterologous systems. LjABCG1 knock-down L. japonicus plants did not show clear phenotypic differences in nodule formation, and not in defense against Pseudomonas syringae, either. In contrast, when LjABCG1 was expressed in the Arabidopsis pdr8-1 mutant, the penetration frequency of Phytophthora infestans, a potato late blight pathogen, was significantly reduced in LjABCG1/pdr8-1 than in pdr8-1 plants. This finding indicated that LjABCG1, at least partially, complemented the phenotype of pdr8 in Arabidopsis, suggesting the multiple roles of this protein in plant-microbe interactions.     

Physicochemical Factors Controlling the Activity and Energy Coupling of an Ionic Strength-gated ATP-binding Cassette (ABC) Transporter

Cells control their volume through the accumulation of compatible solutes. The bacterial ATP-binding cassette transporter OpuA couples compatible solute uptake to ATP hydrolysis. Here, we study the gating mechanism and energy coupling of OpuA reconstituted in lipid nanodiscs. We show that anionic lipids are essential both for the gating and the energy coupling. The tight coupling between substrate binding on extracellular domains and ATP hydrolysis by cytoplasmic nucleotide-binding domains allows the study of transmembrane signaling in nanodiscs. From the tight coupling between processes at opposite sides of the membrane, we infer that the ATPase activity of OpuA in nanodiscs reflects solute translocation. Intriguingly, the substrate-dependent, ionic strength-gated ATPase activity of OpuA in nanodiscs is at least an order of magnitude higher than in lipid vesicles (i.e. with identical membrane lipid composition, ionic strength, and nucleotide and substrate concentrations). Even with the chemical components the same, the lateral pressure (profile) of the nanodiscs will differ from that of the vesicles. We thus propose that membrane tension limits translocation in vesicular systems. Increased macromolecular crowding does not activate OpuA but acts synergistically with ionic strength, presumably by favoring gating interactions of like-charged surfaces via excluded volume effects.     

ABCA1基因多态性与冠心病相关性研究

为了探讨ATP-Binding Cassette Transporter 1（ABCA1）基因R219K多态在中国汉族人群中的分布及其与冠心病（coronary heart disease,CHD）的关系,采用PCR-RFLP方法,对396例CHD患者和417名正常人ABCA1基因R219K多态位点进行分析。结果表明,对照组R219K多态K等位基因及KK基因型的频率（0.465、0.228）较CHD组（0.381、0.162） 显著为高（P＜0.05）;根据发病年龄分组,早发CHD组K等位基因及KK基因型频率（0.34、0.111）明显低于晚发CHD组（0.419、0.205）和对照组（P＜0.05）,而在对照组和晚发CHD组间无此频率差异显著性（P＞0.05）;KK基因型患者血浆甘油三酯（TG）水平较RR基因型显著降低（P＜0.05）;不同基因型患者间血浆高密度脂蛋白胆固醇（HDL-C）水平差异无显著性（P＞0.05）。提示 ABCA1基因R219K多态与CHD存在相关性;KK基因型可能具有对抗动脉粥样硬化的作用,但这种作用不伴有血浆HDL-C水平的改变。     

ATP-Binding Cassette Transporter B4 Anchors the Cell Adhesion Molecule DdCAD-1 to Cell Membrane in Dictyostelium discoideum

In Dictyostelium, soluble cell adhesion molecule, DdCAD-1, regulates cell–cell interaction through an unknown anchoring protein on the plasma membrane. Far western blot analysis using different probes revealed that the potential DdCAD-1 interacting protein was between 64 and 98 kDa. To isolate and identify the anchoring protein, GST-DdCAD-1 and anchoring protein were cross-linked in vivo by chemical cross-linker and stable protein complex was isolated by co-immunoprecipitation assays. The protein cross-linked to DdCAD-1 was extracted from the gel slice and trypsinized. The peptides were subjected to analysis by mass spectrometry, which showed that the putative anchoring protein belongs to ATP-binding cassette transporter family.     

金鱼ABC转运蛋白基因家族成员鉴定及分析

腺苷三磷酸结合盒转运蛋白(ATP-binding cassette transporter,ABC transporter)基因家族在原核生物和真核生物中广泛存在,该家族蛋白能够利用ATP裂解产生的能量将多种底物转运到膜上,参与多种生物过程,如营养摄入、细胞解毒、脂质稳态、信号转导、病毒防御以及抗原呈递等。目前,鱼类中,只在斑马鱼、斑点叉尾鮰和鲤鱼等少数鱼类中对该基因家族进行了系统的研究,关于金鱼ABC转运蛋白基因家族的详细分析,未见报道。本研究中,我们利用三代结合二代测序技术构建的金鱼转录组参考基因集数据,鉴定出55个ABC转运蛋白基因,通过系统进化分析将它们分为8个亚家族(A^H)。即金鱼ABC转运蛋白基因是由10个ABCA、14个ABCB、13个ABCC、5个ABCD、1个ABCE、4个ABCF、7个ABCG和1个ABCH组成。同时,我们将金鱼与斑马鱼、斑点叉尾鮰和鲤鱼等物种ABC转运蛋白基因家族成员的数目进行比较分析,推测硬骨鱼类特异的第3次全基因复制(3R-WGD)和谱系特异的第4次全基因组复制(4R-WGD)对金鱼该基因家族成员数目的影响。本研究结果为金鱼ABC转运蛋白基因功能的研究提供了理论依据。     

Mutant Allele-Specific Uncoupling of PENETRATION3 Functions Reveals Engagement of the ATP-Binding Cassette Transporter in Distinct Tryptophan Metabolic Pathways

Arabidopsis (Arabidopsis thaliana) PENETRATION (PEN) genes quantitatively contribute to the execution of different forms of plant immunity upon challenge with diverse leaf pathogens. PEN3 encodes a plasma membrane-resident pleiotropic drug resistance-type ATP-binding cassette transporter and is thought to act in a pathogen-inducible and PEN2 myrosinase-dependent metabolic pathway in extracellular defense. This metabolic pathway directs the intracellular biosynthesis and activation of tryptophan-derived indole glucosinolates for subsequent PEN3-mediated efflux across the plasma membrane at pathogen contact sites. However, PEN3 also functions in abiotic stress responses to cadmium and indole-3-butyric acid (IBA)-mediated auxin homeostasis in roots, raising the possibility that PEN3 exports multiple functionally unrelated substrates. Here, we describe the isolation of a pen3 allele, designated pen3-5, that encodes a dysfunctional protein that accumulates in planta like wild-type PEN3. The specific mutation in pen3-5 uncouples PEN3 functions in IBA-stimulated root growth modulation, callose deposition induced with a conserved peptide epitope of bacterial flagellin (flg22), and pathogen-inducible salicylic acid accumulation from PEN3 activity in extracellular defense, indicating the engagement of multiple PEN3 substrates in different PEN3-dependent biological processes. We identified 4-O-β-d-glucosyl-indol-3-yl formamide (4OGlcI3F) as a pathogen-inducible, tryptophan-derived compound that overaccumulates in pen3 leaf tissue and has biosynthesis that is dependent on an intact PEN2 metabolic pathway. We propose that a precursor of 4OGlcI3F is the PEN3 substrate in extracellular pathogen defense. These precursors, the shared indole core present in IBA and 4OGlcI3F, and allele-specific uncoupling of a subset of PEN3 functions suggest that PEN3 transports distinct indole-type metabolites in distinct biological processes.The ATP-binding cassette (ABC) transporters constitute one of the largest protein families in the plant kingdom (Rea, 2007; Verrier et al., 2008; Kang et al., 2011). They share a core structure comprising highly conserved nucleotide-binding domains (NBDs) and transmembrane domains (TMDs). In the model plant Arabidopsis (Arabidopsis thaliana), there are over 120 ABC transporters grouped into 13 subfamilies classified by NBD phylogeny, the length of the protein, and/or the organization of the domains (Verrier et al., 2008). These transmembrane proteins play important roles in plant development, organ formation, and plant response to abiotic and biotic stresses (Kang et al., 2011). Known substrates translocated by characterized ABC transporters cover a range of small molecules, including abscisic acid, auxin and/or auxin precursors (Lin and Wang, 2005; Lewis et al., 2007; Wu et al., 2007), phytochelatin, glutathione and/or glutathione conjugates (Liu et al., 2001; Song et al., 2010), folates and folate homologs (Klein et al., 2004; Raichaudhuri et al., 2009), and many other molecules.Pleiotropic drug resistance (PDR)-type full-size ABC transporters belong to the ABC transporter protein subfamily G (ABCG) and are found exclusively in fungi and plants (Verrier et al., 2008; Kang et al., 2011). The expression of plant genes encoding PDR proteins is often stimulated by microbial infection and defense phytohormones, such as salicylic acid (SA) and jasmonic acid. For example, tobacco (Nicotiana tabacum) PDR1, which is induced by Phytophthora infestans elicitins, flagellin, and methyl jasmonate, directly transports diterpenes in tobacco Bright Yellow-2 suspension cells (Sasabe et al., 2002; Crouzet et al., 2013). NtPDR5 is induced by methyl jasmonate and wounding and plays a role in herbivore resistance (Bienert et al., 2012). Nicotiana plumbaginifolia PDR1 transports diterpene sclareol and is induced by pathogen colonization or jasmonic acid treatment (Stukkens et al., 2005). Wheat (Triticum aestivum) PDR transporter LEAF RUST RESISTANCE34 confers durable, race-nonspecific resistance to multiple fungal pathogens, and the corresponding gene is highly relevant in breeding disease-resistant wheat cultivars (Krattinger et al., 2009). PDR transporters are not only involved in plant defense to pathogenic microorganisms. Petunia hybrida PDR1 is a strigolactone exporter critical for the establishment of symbiotic interactions with arbuscular mycorrhizal fungi (Kretzschmar et al., 2012). Arabidopsis pdr2 plants revealed drastic changes in root exudate profiles, and the composition of root-associated bacterial communities seems to be altered in the mutant plants (Badri et al., 2008, 2009).The Arabidopsis PENETRATION3 (PEN3)/PDR8/ABCG36 PDR-type ABC transporter is unusual, because it has been functionally assigned to several biotic and abiotic stress responses as well as in the transport of the auxin precursor indole-3-butyric acid (IBA). Mutant pen3 plants are defective in extracellular (apoplastic) defense to nonadapted powdery mildew pathogens, including Blumeria graminis and Erysiphe pisi, and the nonadapted oomycete pathogen P. infestans (Stein et al., 2006). Genetic screens for impaired extracellular defense in nonhost resistance to the nonadapted powdery mildew pathogens also identified SYNTAXIN RELATED PROTEIN1 (SYR1), also known as SYP121/PEN1 and PEN2, which encode a plasma membrane-resident syntaxin and a myrosinase, respectively (Leyman et al., 1999; Collins et al., 2003; Lipka et al., 2005; Stein et al., 2006). PEN2 and PEN3 act in the same pathway for extracellular defense, whereas PEN1 functions in a parallel secretory defense pathway (Collins et al., 2003; Kwon et al., 2008; Kim et al., 2014). PEN2 function has been assigned to a glucosinolate metabolic pathway, which comprises biosynthesis of indole glucosinolates (IGs), pathogen-inducible redirection of this biosynthesis through the CYTOCHROME P450 81F2 (CYP81F2) monooxygenase to 4-methoxyindol-3-ylmethylglucosinolate (4MI3G), and 4MI3G activation by PEN2 myrosinase through deglucosylation (Bednarek et al., 2009). Additional metabolized PEN2 products are thought to be exported to the apoplast at pathogen contact sites by plasma membrane-resident PEN3/PDR8/ABCG36 (Stein et al., 2006; Bednarek et al., 2009), but the structures of PEN3 substrates for extracellular defense remain to be identified.PEN genes were originally identified as components of nonhost resistance to nonadapted pathogens. This type of general plant immunity can be triggered upon perception of evolutionary conserved microbe-associated molecular patterns (MAMPs) by membrane-resident pattern recognition receptors (PRRs) or upon activation of intracellular nucleotide-binding and Leu-rich repeat (NLR)-type immune receptors that detect the presence of race-specific pathogen effectors (Schulze-Lefert and Panstruga, 2011). Recently, PEN1, PEN2, and PEN3 were shown to contribute quantitatively to race-specific immune responses against host-adapted bacterial and oomycete pathogens after immune response activation by intracellular NLR-type immune receptors (Johansson et al., 2014). This genetic evidence and known biochemical PEN activities strongly suggest PEN engagements in the execution of plant immune responses triggered by both PRR- and NLR-type immune receptors. Although much is known about plant immune receptors recognizing nonself molecules and subsequent phytohormone-dependent defense signaling, the molecules that execute immune responses to restrict pathogen growth remain largely unknown. In this context, pen mutants are useful tools to identify candidate molecules or compound classes contributing to defense response execution.pen3 null mutant phenotypes include an enhanced disease resistance (edr) to the host-adapted powdery mildew Golovinomyces cichoracearum (formerly Erysiphe cichoracearum), and this infection phenotype is dependent on SA biosynthesis but not dependent on PEN2 (Kobae et al., 2006; Stein et al., 2006). This indicates separable PEN3 functions in extracellular defense to nonadapted powdery mildews and for host colonization by host-adapted G. cichoracearum. PEN2 and PEN3 also act together to limit growth of both host-adapted and nonadapted pathogenic strains of the necrotrophic fungus Plectosphaerella cucumerina. However, pen3 plants are more susceptible to P. cucumerina infection than pen2 plants, suggesting yet another PEN2-independent function of PEN3 in defense of necrotrophic pathogens (Stein et al., 2006; Sanchez-Vallet et al., 2010).The function of PEN3 is not restricted to the innate immune system of Arabidopsis. An excised root tip auxin transport assay showed that root tips of pen3 hyperaccumulate [³H]IBA, suggesting that IBA is a PEN3 substrate. Similarly, a leaf protoplast Cd transport assay has shown that ¹⁰⁹Cd levels are higher in AtPEN3 RNA interference plants and lower in overexpressing plants compared with the wild type, indicating that PEN3 directly transports Cd²⁺ or its conjugates (Kim et al., 2007; Strader and Bartel, 2009; Ruzicka et al., 2010). Together, these data suggest additional PEN3 functions in IBA-mediated auxin homeostasis and cadmium tolerance.In this study, we have isolated and characterized the pen3-5 allele associated with a single-amino acid substitution. The mutant protein, unlike most previously isolated pen3 alleles, accumulates in planta like wild-type PEN3. Using this mutant and all other previously described pen3 alleles, we show an allele-specific uncoupling of a subset of PEN3 functions. We then applied metabolic profiling of pathogen-inoculated wild-type and pen3 plants to identify pathogen-inducible compounds that hyperaccumulate in pen3 leaf tissue. Purification and mass spectrometry of a prominent hyperaccumulating compound identified an indole derivative 4-O-β-d-glucosyl-indol-3-yl formamide (4OGlcI3F), whose biosynthesis is dependent on the PEN2 metabolic pathway. We propose that one or several precursors of this molecule serve as the PEN3 substrate(s) for export into the apoplast during extracellular defense.     

Retinoid Binding Properties of Nucleotide Binding Domain 1 of the Stargardt Disease-associated ATP Binding Cassette (ABC) Transporter,ABCA4

The retina-specific ATP binding cassette transporter, ABCA4 protein, is associated with a broad range of inherited macular degenerations, including Stargardt disease, autosomal recessive cone rod dystrophy, and fundus flavimaculatus. In order to understand its role in retinal transport in rod out segment discs, we have investigated the interactions of the soluble domains of ABCA4 with both 11-cis- and all-trans-retinal. Using fluorescence anisotropy-based binding analysis and recombinant polypeptides derived from the amino acid sequences of the four soluble domains of ABCA4, we demonstrated that the nucleotide binding domain 1 (NBD1) specifically bound 11-cis-retinal. Its affinity for all-trans-retinal was markedly reduced. Stargardt disease-associated mutations in this domain resulted in attenuation of 11-cis-retinal binding. Significant differences in 11-cis-retinal binding affinities were observed between NBD1 and other cytoplasmic and lumenal domains of ABCA4. The results suggest a possible role of ABCA4 and, in particular, the NBD1 domain in 11-cis-retinal binding. These results also correlate well with a recent report on the in vivo role of ABCA4 in 11-cis-retinal transport.