Multidrug And Toxin Extrusion Research Articles

Overexpression of BdMATE Gene Improves Aluminum Tolerance in Setaria viridis.

Acidic soils are distributed worldwide, predominantly in tropical and subtropical areas, reaching around 50% of the arable soil. This type of soil strongly reduces crop production, mainly because of the presence of aluminum, which has its solubility increased at low pH levels. A well-known physiological mechanism used by plants to cope with Al stress involves activation of membrane transporters responsible for organic acid anions secretion from the root apex to the rhizosphere, which chelate Al, preventing its absorption by roots. In sorghum, a membrane transporter gene belonging to multidrug and toxic compound extrusion (MATE) family was identified and characterized as an aluminum-activated citrate transporter gene responsible for Al tolerance in this crop. Setaria viridis is an emerging model for C4 species and it is an important model to validate some genes for further C4 crops transformation, such as sugarcane, maize, and wheat. In the present work, Setaria viridis was used as a model plant to overexpress a newly identified MATE gene from Brachypodium distachyon (BdMATE), closely related to SbMATE, for aluminum tolerance assays. Transgenic S. viridis plants overexpressing a BdMATE presented an improved Al tolerance phenotype, characterized by sustained root growth and exclusion of aluminum from the root apex in transgenic plants, as confirmed by hematoxylin assay. In addition, transgenic plants showed higher root citrate exudation into the rhizosphere, suggesting that Al tolerance improvement in these plants could be related to the chelation of the metal by the organic acid anion. These results suggest that BdMATE gene can be used to transform C4 crops of economic importance with improved aluminum tolerance.

Site-specific His/Asp phosphoproteomic analysis of prokaryotes reveals putative targets for drug resistance

BackgroundPhosphorylation of amino acid residues on proteins is an important and common post-translational modification in both eukaryotes and prokaryotes. Most research work has been focused on phosphorylation of serine, threonine or tyrosine residues, whereas phosphorylation of other amino acids are significantly less clear due to the controversy on their stability under standard bioanalytical conditions.ResultsHere we applied a shotgun strategy to analyze the histidine and aspartate phosphorylations in different microbes. Our results collectively indicate that histidine and aspartate phosphorylations frequently occur also in proteins that are not part of the two-component systems. Noticeably, a number of the modified proteins are pathogenesis-related or essential for survival in host. These include the zinc ion periplasmic transporter ZnuA in Acinetobacter baumannii SK17, the multidrug and toxic compound extrusion (MATE) channel YeeO in Klebsiella pneumoniae NTUH-K2044, branched amino acid transporter AzlC in Vibrio vulnificus and the RNA-modifying pseudouridine synthase in Helicobacter pylori.ConclusionsIn summary, histidine and aspartate phosphorylation is likely to be ubiquitous and to take place in proteins of various functions. This work also sheds light into how these functionally important proteins and potential drug targets might be regulated at a post-translational level.

Involvement of the Multidrug and Toxic Compound Extrusion Transporter in Testosterone Release from Cultured Pig Leydig Cells

Testosterone is considered to be released from Leydig cells via passive diffusion because of its hydrophobicity; however, the exact mechanism underlying testosterone secretion and the transporter involved are both unknown. Multidrug and toxic compound extrusion (MATE) transporters are predominantly found in the kidneys and liver and are thought to function in the elimination of metabolic organic cations during the final step of excretion in the kidney. In contrast, mMATE2 has been shown to be predominantly expressed in testicular Leydig cells. Although the physiological function of mMATE2 in Leydig cells is unknown, we hypothesized that mMATE2 acts as a testosterone exporter and is responsible for the secretion of testosterone from Leydig cells. Therefore, in the present study, we investigated the involvement of the MATE transporter in testosterone secretion from pig Leydig cells. Immunohistochemical analysis with anti-pig MATE2 antiserum indicated that the MATE transporter is present in pig Leydig cells. Additionally, treatment with the MATE inhibitors cimetidine and pyrimethamine reduced the testosterone secretion from pig Leydig cells but increased the intracellular testosterone levels. Estradiol release and intracellular estradiol level induced by human chorionic gonadotropin (hCG) further increased with cimetidine treatment. These results indicated that testosterone produced by hCG treatment is secreted from Leydig cells via the MATE transporter; however, in the presence of cimetidine or pyrimethamine, this MATE transporter-mediated secretion was inhibited, resulting in increased intracellular testosterone levels and estradiol production in Leydig cells. Thus, the MATE transporter may be responsible for testosterone secretion from Leydig cells.

Interaction and Transport of Methamphetamine and its Primary Metabolites by Organic Cation and Multidrug and Toxin Extrusion Transporters

Methamphetamine is one of the most abused illicit drugs with roughly 1.2 million users in the United States alone. A large portion of methamphetamine and its metabolites is eliminated by the kidney with renal clearance larger than glomerular filtration clearance. Yet the mechanism of active renal secretion is poorly understood. The goals of this study were to characterize the interaction of methamphetamine and its major metabolites with organic cation transporters (OCTs) and multidrug and toxin extrusion (MATE) transporters and to identify the major transporters involved in the disposition of methamphetamine and its major metabolites, amphetamine and para-hydroxymethamphetamine (p-OHMA). We used cell lines stably expressing relevant transporters to show that methamphetamine and its metabolites inhibit human OCTs 1-3 (hOCT1-3) and hMATE1/2-K with the greatest potencies against hOCT1 and hOCT2. Methamphetamine and amphetamine are substrates of hOCT2, hMATE1, and hMATE2-K, but not hOCT1 and hOCT3. p-OHMA is transported by hOCT1-3 and hMATE1, but not hMATE2-K. In contrast, organic anion transporters 1 and 3 do not interact with or transport these compounds. Methamphetamine and its metabolites exhibited complex interactions with hOCT1 and hOCT2, suggesting the existence of multiple binding sites. Our studies suggest the involvement of the renal OCT2/MATE pathway in tubular secretion of methamphetamine and its major metabolites and the potential of drug-drug interactions with substrates or inhibitors of the OCTs. This information may be considered when prescribing medications to suspected or known abusers of methamphetamine to mitigate the risk of increased toxicity or reduced therapeutic efficacy.

Diverse functions of multidrug and toxin extrusion (MATE) transporters in citric acid efflux and metal homeostasis in Medicago truncatula.

The multidrug and toxin extrusion (MATE) transporter family comprises 70 members in the Medicago truncatula genome, and they play seemingly important, yet mostly uncharacterized, physiological functions. Here, we employed bioinformatics and molecular genetics to identify and characterize MATE transporters involved in citric acid export, Al3+ tolerance and Fe translocation. MtMATE69 is a citric acid transporter induced by Fe-deficiency. Overexpression of MtMATE69 in hairy roots altered Fe homeostasis and hormone levels under Fe-deficient or Fe-oversupplied conditions. MtMATE66 is a plasma membrane citric acid transporter primarily expressed in root epidermal cells. The mtmate66 mutant had less root growth than the wild type under Al3+ stress, and seedlings were chlorotic under Fe-deficient conditions. Overexpression of MtMATE66 rendered hairy roots more tolerant to Al3+ toxicity. MtMATE55 is involved in seedling development and iron homeostasis, as well as hormone signaling. The mtmate55 mutant had delayed development and chlorotic leaves in mature plants. Both knock-out and overexpression mutants of MtMATE55 showed altered Fe accumulation and abnormal hormone levels compared with the wild type. We demonstrate that the zinc-finger transcription factor MtSTOP is essentially required for MtMATE66 expression and plant resistance to H+ and Al3+ toxicity. The proper expression of two previously characterized MATE flavonoid transporters MtMATE1 and MtMATE2 also depends on several transcription factors. This study reveals not only functional diversity of MATE transporters and regulatory mechanisms in legumes against H+ and Al3+ stresses, but also casts light on their role in metal nutrition and hormone signaling under various stresses.

Renal Drug Transporters and Drug Interactions.

Transporters in proximal renal tubules contribute to the disposition of numerous drugs. Furthermore, the molecular mechanisms of tubular secretion have been progressively elucidated during the past decades. Organic anions tend to be secreted by the transport proteins OAT1, OAT3 and OATP4C1 on the basolateral side of tubular cells, and multidrug resistance protein (MRP)2, MRP4, OATP1A2 and breast cancer resistance protein (BCRP) on the apical side. Organic cations are secreted by organic cation transporter (OCT)2 on the basolateral side, and multidrug and toxic compound extrusion (MATE) proteins MATE1, MATE2/2-K, P-glycoprotein, organic cation and carnitine transporter (OCTN)1 and OCTN2 on the apical side. Significant drug-drug interactions (DDIs) may affect any of these transporters, altering the clearance and, consequently, the efficacy and/or toxicity of substrate drugs. Interactions at the level of basolateral transporters typically decrease the clearance of the victim drug, causing higher systemic exposure. Interactions at the apical level can also lower drug clearance, but may be associated with higher renal toxicity, due to intracellular accumulation. Whereas the importance of glomerular filtration in drug disposition is largely appreciated among clinicians, DDIs involving renal transporters are less well recognized. This review summarizes current knowledge on the roles, quantitative importance and clinical relevance of these transporters in drug therapy. It proposes an approach based on substrate-inhibitor associations for predicting potential tubular-based DDIs and preventing their adverse consequences. We provide a comprehensive list of known drug interactions with renally-expressed transporters. While many of these interactions have limited clinical consequences, some involving high-risk drugs (e.g. methotrexate) definitely deserve the attention of prescribers.

Two tonoplast MATE proteins function as turgor-regulating chloride channels in Arabidopsis

The central vacuole in a plant cell occupies the majority of the cellular volume and plays a key role in turgor regulation. The vacuolar membrane (tonoplast) contains a large number of transporters that mediate fluxes of solutes and water, thereby adjusting cell turgor in response to developmental and environmental signals. We report that two tonoplast Detoxification efflux carrier (DTX)/Multidrug and Toxic Compound Extrusion (MATE) transporters, DTX33 and DTX35, function as chloride channels essential for turgor regulation in Arabidopsis Ectopic expression of each transporter in Nicotiana benthamiana mesophyll cells elicited a large voltage-dependent inward chloride current across the tonoplast, showing that DTX33 and DTX35 each constitute a functional channel. Both channels are highly expressed in Arabidopsis tissues, including root hairs and guard cells that experience rapid turgor changes during root-hair elongation and stomatal movements. Disruption of these two genes, either in single or double mutants, resulted in shorter root hairs and smaller stomatal aperture, with double mutants showing more severe defects, suggesting that these two channels function additively to facilitate anion influx into the vacuole during cell expansion. In addition, dtx35 single mutant showed lower fertility as a result of a defect in pollen-tube growth. Indeed, patch-clamp recording of isolated vacuoles indicated that the inward chloride channel activity across the tonoplast was impaired in the double mutant. Because MATE proteins are widely known transporters of organic compounds, finding MATE members as chloride channels expands the functional definition of this large family of transporters.

MATE2 Expression Is Associated with Cancer Cell Response to Metformin.

BackgroundThere is great interest in repurposing the commonly prescribed anti-diabetic drug metformin for cancer therapy. Intracellular uptake and retention of metformin is affected by the expression of organic cation transporters (OCT) 1–3 and by multidrug and toxic compound extrusion (MATE) 1–2. Inside cells, metformin inhibits mitochondrial function, which leads to reduced oxygen consumption and inhibition of proliferation. Reduced oxygen consumption can lead to improved tumor oxygenation and radiation response.PurposeHere we sought to determine if there is an association between the effects of metformin on inhibiting oxygen consumption, proliferation and expression of OCTs and MATEs in a panel of 19 cancer cell lines.ResultsThere was relatively large variability in the anti-proliferative response of different cell lines to metformin, with a subset of cell lines being very resistant. In contrast, all cell lines demonstrated sensitivity to the inhibition of oxygen consumption by metformin, with relatively small variation. The expression of OCT1 correlated with expression of both OCT2 and OCT3. OCT1 and OCT2 were relatively uniformly expressed, whereas expression of OCT3, MATE1 and MATE2 showed substantial variation across lines. There were statistically significant associations between resistance to inhibition of proliferation and MATE2 expression, as well as between sensitivity to inhibition of oxygen consumption and OCT3 expression. One cell line (LNCaP) with high OCT3 and low MATE2 expression in concert, had substantially higher intracellular metformin concentration than other cell lines, and was exquisitely sensitive to both anti-proliferative and anti-respiratory effects. In all other cell lines, the concentration of metformin required to inhibit oxygen consumption acutely in vitro was substantially higher than that achieved in the plasma of diabetic patients. However, administering anti-diabetic doses of metformin to tumor-bearing mice resulted in intratumoral accumulation of metformin and reduced hypoxic tumor fractions.ConclusionsAll cancer cells are susceptible to inhibition of oxygen consumption by metformin, which results in reduced hypoxic tumor fractions beneficial for the response to radiotherapy. High MATE2 expression may result in resistance to the anti-proliferative effect of metformin and should be considered as a negative predictive biomarker in clinical trials.

The biosynthetic gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor contains its co-expressed vacuolar MATE transporter.

Genomic gene clusters for the biosynthesis of chemical defence compounds are increasingly identified in plant genomes. We previously reported the independent evolution of biosynthetic gene clusters for cyanogenic glucoside biosynthesis in three plant lineages. Here we report that the gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor additionally contains a gene, SbMATE2, encoding a transporter of the multidrug and toxic compound extrusion (MATE) family, which is co-expressed with the biosynthetic genes. The predicted localisation of SbMATE2 to the vacuolar membrane was demonstrated experimentally by transient expression of a SbMATE2-YFP fusion protein and confocal microscopy. Transport studies in Xenopus laevis oocytes demonstrate that SbMATE2 is able to transport dhurrin. In addition, SbMATE2 was able to transport non-endogenous cyanogenic glucosides, but not the anthocyanin cyanidin 3-O-glucoside or the glucosinolate indol-3-yl-methyl glucosinolate. The genomic co-localisation of a transporter gene with the biosynthetic genes producing the transported compound is discussed in relation to the role self-toxicity of chemical defence compounds may play in the formation of gene clusters.

The similar and different evolutionary trends of MATE family occurred between rice and Arabidopsis thaliana.

BackgroundMultidrug and toxic compound extrusion (MATE) transporter proteins are present in all organisms. Although the functions of some MATE gene family members have been studied in plants, few studies have investigated the gene expansion patterns, functional divergence, or the effects of positive selection.ResultsForty-five MATE genes from rice and 56 from Arabidopsis were identified and grouped into four subfamilies. MATE family genes have similar exon-intron structures in rice and Arabidopsis; MATE gene structures are conserved in each subfamily but differ among subfamilies. In both species, the MATE gene family has expanded mainly through tandem and segmental duplications. A transcriptome atlas showed considerable differences in expression among the genes, in terms of transcript abundance and expression patterns under normal growth conditions, indicating wide functional divergence in this family. In both rice and Arabidopsis, the MATE genes showed consistent functional divergence trends, with highly significant Type-I divergence in each subfamily, while Type-II divergence mainly occurred in subfamily III. The Type-II coefficients between rice subfamilies I/III, II/III, and IV/III were all significantly greater than zero, while only the Type-II coefficient between Arabidopsis IV/III subfamilies was significantly greater than zero.A site-specific model analysis indicated that MATE genes have relatively conserved evolutionary trends. A branch-site model suggested that the extent of positive selection on each subfamily of rice and Arabidopsis was different: subfamily II of Arabidopsis showed higher positive selection than other subfamilies, whereas in rice, positive selection was highest in subfamily III. In addition, the analyses identified 18 rice sites and 7 Arabidopsis sites that were responsible for positive selection and for Type-I and Type-II functional divergence; there were no common sites between rice and Arabidopsis. Five coevolving amino acid sites were identified in rice and three in Arabidopsis; these sites might have important roles in maintaining local structural stability and protein functional domains.ConclusionsWe demonstrate that the MATE gene family expanded through tandem and segmental duplication in both rice and Arabidopsis. Overall, the results of our analyses contribute to improved understanding of the molecular evolution and functions of the MATE gene family in plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0895-0) contains supplementary material, which is available to authorized users.

Indole-3-acetic acid in plant–pathogen interactions: a key molecule for in planta bacterial virulence and fitness

The plant pathogenic bacterium Pseudomonas savastanoi, the causal agent of olive and oleander knot disease, uses the so-called “indole-3-acetamide pathway” to convert tryptophan to indole-3-acetic acid (IAA) via a two-step pathway catalyzed by enzymes encoded by the genes in the iaaM/iaaH operon. Moreover, pathovar nerii of P. savastanoi is able to conjugate IAA to lysine to generate the less biologically active compound IAA-Lys via the enzyme IAA-lysine synthase encoded by the iaaL gene. Interestingly, iaaL is now known to be widespread in many Pseudomonas syringae pathovars, even in the absence of the iaaM and iaaH genes for IAA biosynthesis.Here, two knockout mutants, ΔiaaL and ΔiaaM, of strain Psn23 of P. savastanoi pv. nerii were produced. Pathogenicity tests using the host plant Nerium oleander showed that ΔiaaL and ΔiaaM were hypervirulent and hypovirulent, respectively and these features appeared to be related to their differential production of free IAA. Using the Phenotype Microarray approach, the chemical sensitivity of these mutants was shown to be comparable to that of wild-type Psn23. The main exception was 8 hydroxyquinoline, a toxic compound that is naturally present in plant exudates and is used as a biocide, which severely impaired the growth of ΔiaaL and ΔiaaM, as well as growth of the non-pathogenic mutant ΔhrpA, which lacks a functional Type Three Secretion System (TTSS). According to bioinformatics analysis of the Psn23 genome, a gene encoding a putative Multidrug and Toxic compound Extrusion (MATE) transporter, was found upstream of iaaL. Similarly to iaaL and iaaM, its expression appeared to be TTSS-dependent. Moreover, auxin-responsive elements were identified for the first time in the modular promoters of both the iaaL gene and the iaaM/iaaH operon of P. savastanoi, suggesting their IAA-inducible transcription. Gene expression analysis of several genes related to TTSS, IAA metabolism and drug resistance confirmed the presence of a concerted regulatory network in this phytopathogen among virulence, fitness and drug efflux.

Insoluble-Bound Phenolics in Food.

This contribution provides a review of the topic of insoluble-bound phenolics, especially their localization, synthesis, transfer and formation in plant cells, as well as their metabolism in the human digestive system and corresponding bioactivities. In addition, their release from the food matrix during food processing and extraction methods are discussed. The synthesis of phenolics takes place mainly at the endoplasmic reticulum and they are then transferred to each organ through transport proteins such as the ATP-binding cassette (ABC) and multidrug and toxic compound extrusion (MATE) transporter at the organ’s compartment membrane or via transport vesicles such as cytoplasmic and Golgi vesicles, leading to the formation of soluble and insoluble-bound phenolics at the vacuole and cell wall matrix, respectively. This part has not been adequately discussed in the food science literature, especially regarding the synthesis site and their transfer at the cellular level, thus this contribution provides valuable information to the involved scientists. The bound phenolics cannot be absorbed at the small intestine as the soluble phenolics do (5%–10%), thus passing into the large intestine and undergoing fermentation by a number of microorganisms, partially released from cell wall matrix of foods. Bound phenolics such as phenolic acids and flavonoids display strong bioactivities such as anticancer, anti-inflammation and cardiovascular disease ameliorating effects. They can be extracted by several methods such as acid, alkali and enzymatic hydrolysis to quantify their contents in foods. In addition, they can also be released from the cell wall matrix during food processing procedures such as fermentation, germination, roasting, extrusion cooking and boiling. This review provides critical information for better understanding the insoluble-bound phenolics in food and fills an existing gap in the literature.

Associations of genetic polymorphisms of the transporters organic cation transporter 2 (OCT2), multidrug and toxin extrusion 1 (MATE1), and ATP-binding cassette subfamily C member 2 (ABCC2) with platinum-based chemotherapy response and toxicity in non-small cell lung cancer patients.

BackgroundPlatinum-based chemotherapy is the first-line treatment of non-small cell lung cancer (NSCLC); it is therefore important to discover biomarkers that can be used to predict the efficacy and toxicity of this treatment. Four important transporter genes are expressed in the kidney, including organic cation transporter 2 (OCT2), multidrug and toxin extrusion 1 (MATE1), ATP-binding cassette subfamily B member 1 (ABCB1), and ATP-binding cassette subfamily C member 2 (ABCC2), and genetic polymorphisms in these genes may alter the efficacy and adverse effects of platinum drugs. This study aimed to evaluate the association of genetic polymorphisms of these transporters with platinum-based chemotherapy response and toxicity in NSCLC patients.MethodsA total of 403 Chinese NSCLC patients were recruited for this study. All patients were newly diagnosed with NSCLC and received at least two cycles of platinum-based chemotherapy. The tumor response and toxicity were evaluated after two cycles of treatment, and the patients’ genomic DNA was extracted. Seven single-nucleotide polymorphisms in four transporter genes were selected to investigate their associations with platinum-based chemotherapy toxicity and response.ResultsOCT2 rs316019 was associated with hepatotoxicity (P = 0.026) and hematological toxicity (P = 0.039), and MATE1 rs2289669 was associated with hematological toxicity induced by platinum (P = 0.016). In addition, ABCC2 rs717620 was significantly associated with the platinum-based chemotherapy response (P = 0.031). ABCB1 polymorphisms were associated with neither response nor toxicity.ConclusionOCT2 rs316019, MATE1 rs2289669, and ABCC2 rs717620 might be potential clinical markers for predicting chemotherapy toxicity and response induced by platinum-based treatment in NSCLC patients.Trial registration Chinese Clinical Trial Registry ChiCTR-RNC-12002892

Genomewide analysis of MATE-type gene family in maize reveals microsynteny and their expression patterns under aluminum treatment.

Multidrug and toxic compound extrusion (MATE) proteins are a group of secondary active transporters, which widely exist in all living organisms and play important role in the detoxication of endogenous secondary metabolites and exogenous agents. However, to date, no systematic and comprehensive study of this family is reported in maize. Here, a total of 49 MATE genes (ZmMATE) were identified and divided into seven groups by phylogenetic analysis. Conserved intro-exon structures and motif compositions were investigated in these genes. Results by gene locations indicated that these genes were unevenly distributed among all 10 chromosomes. Tandem and segmental duplications appeared to contribute to the expansion and evolution of this gene family. The Ka/Ks ratios suggested that the ZmMATE has undergone large-scale purifying selection on the maize genome. Interspecies microsynteny analysis revealed that there were independent gene duplication events of 10 ZmMATE. In addition, most maize MATE genes exhibited different expression profiles in diverse tissues and developmental stages. Sixteen MATE genes were chosen for further quantitative real-time polymerase chain reaction analysis showed differential expression patterns in response to aluminum treatment. These results provide a useful clue for future studies on the identification of MATE genes and functional analysis of MATE proteins in maize.

Gen dan QTL Pengendali Toleransi Tanaman terhadap Keracunan Aluminium dan Aplikasinya untuk Pemuliaan Tanaman di Indonesia

<p>Genetic knowledge of loci controlling Al toxicity tolerance is the key for a successful breeding program in developing Al<br />tolerant cultivars. Tolerance level of crop plants to Al toxicity is genetically controlled. The gene inheritance pattern is mainly<br />resulted from intensive studies of cereal crops, such as wheat, sorghum, maize, and rice. The trait can be controlled by a<br />single dominant gene, a single dominant gene with many alleles, a pair of dominant genes, or by many genes (QTL). The<br />majority of the Al tolerance genes identified so far belongs to two independent groups of gene families, i.e. aluminumactivated<br />malate transporter (ALMT) and multidrug and toxic compound extrusion (MATE), both encoding transport proteins<br />involved in Al-activated organic acid release, mainly citrate and malate. The variations in Al toxicity tolerance phenotypes are<br />strongly correlated with the expressions of such genes in the root apical cells. Many Al tolerance QTLs have been mapped in<br />the genomes of various crop species and were found to be colocated with the ALMT and MATE genes. The genetic maps of<br />the Al tolerance genes and QTLs facilitate breeding programs for developing Al-tolerant cultivars through marker-assisted<br />breeding methods. Al tolerance genes that have been isolated from genetically unrelated species can be used in genetic<br />transformation studies of crop genotypes sexually incompatible to the gene source genotypes. The application of these<br />molecular breeding methods expedites breeding programs to develop crop cultivars tolerance to Al toxicity and acid soils.<br />Genomic technologies by using next-generation sequencing and high-throughput genotyping system accelerate Al toxicity<br />tolerance gene and QTL discoveries of various crop species. The modern genomic technologies also facilitate more<br />comprehensive PGR characterization and utilization to accelerate identification and isolation of the Al tolerance genes and<br />QTLs to be used in a more comprehensive breeding program to support national food self sufficiency and food security<br />programs.</p>

Urinary Dopamine as a Potential Index of the Transport Activity of Multidrug and Toxin Extrusion in the Kidney.

Dopamine is a cationic natriuretic catecholamine synthesized in proximal tubular cells (PTCs) of the kidney before secretion into the lumen, a key site of its action. However, the molecular mechanisms underlying dopamine secretion into the lumen remain unclear. Multidrug and toxin extrusion (MATE) is a H+/organic cation antiporter that is highly expressed in the brush border membrane of PTCs and mediates the efflux of organic cations, including metformin and cisplatin, from the epithelial cells into the urine. Therefore, we hypothesized that MATE mediates dopamine secretion, a cationic catecholamine, into the tubule lumen, thereby regulating natriuresis. Here, we show that [3H]dopamine uptake in human (h) MATE1-, hMATE-2K- and mouse (m) MATE-expressing cells exhibited saturable kinetics. Fluid retention and decreased urinary excretion of dopamine and Na+ were observed in Mate1-knockout mice compared to that in wild-type mice. Imatinib, a MATE inhibitor, inhibited [3H]dopamine uptake by hMATE1-, hMATE2-K- and mMATE1-expressing cells in a concentration-dependent manner. At clinically-relevant concentrations, imatinib inhibited [3H]dopamine uptake by hMATE1- and hMATE2-K-expressing cells. The urinary excretion of dopamine and Na+ decreased and fluid retention occurred in imatinib-treated mice. In conclusion, MATE transporters secrete renally-synthesized dopamine, and therefore, urinary dopamine has the potential to be an index of the MATE transporter activity.

Molecular cloning and functional characterization of DkMATE1 involved in proanthocyanidin precursor transport in persimmon (Diospyros kaki Thunb.) fruit

Persimmon fruits accumulate a large amount of proanthocyanidins (PAs) in “tannin cells” during development that cause the sensation of astringency due to coagulation of oral proteins. Pollination-constant non-astringent (PCNA) is a spontaneous mutant persimmon phenotype that loses its astringency naturally on the tree at maturity; while the more common non-PCNA fruits remain rich in PAs until they are fully ripened. Here, we isolated a DkMATE1 gene encoding a Multidrug And Toxic Compound Extrusion (MATE) family protein from the Chinese PCNA (C-PCNA) ‘Eshi 1’. Expression patterns of DkMATE1 were positively correlated with the accumulation of PAs in different types of persimmons fruits during fruit development. An analysis of the inferred amino acid sequences and phylogenetic relationships indicated that DkMATE1 is a putative PA precursor transporter, and subcellular localization assays revealed that DkMATE1 is localized in the vacuolar membrane. Ectopic expression of the DkMATE1 in Arabidopsis tt12 mutant supported that DkMATE1 could complement its biological function in transporting epicatechin 3′-O-glucoside as a PAs precursor from the cytoplasm to vacuole. Furthermore, the transient over-expression and silencing of DkMATE1 in ‘Mopanshi’ persimmon leaves resulted in a significant increase and a decrease in PA content, respectively. The analysis of cis-elements in DkMATE1 promoter regions indicated that DkMATE1 might be regulated by DkMYB4, another well-known structural gene in persimmon. Overall, our results show that DkMATE1 may be an essential PA precursor membrane transporter that plays an important role in PA biosynthesis in persimmon.

Role of OCT2 and MATE1 in renal disposition and toxicity of nitidine chloride.

Nitidine chloride (NC), a benzophenanthridine alkaloid, has various biological properties including anticancer and analgesic activities. The aim of the present study was to evaluate the role of organic cation transporter 2 (OCT2) and multidrug and toxin extrusion 1 (MATE1) in the renal disposition and nephrotoxicity of NC. MDCK cells stably expressing human OCT2 and/or hMATE1 were used to investigate the OCT2- and MATE1-mediated transport of NC. In addition, the accumulation of NC and its potential toxicity were studied in rat primary-cultured proximal tubular (rPCPT) cells and in rats in vivo. NC was found to be a high-affinity substrate of both OCT2 and MATE1 with high cytotoxicity in MDCK-hOCT2/hMATE1 and MDCK-hOCT2 compared to mock cells. The OCT2 inhibitors, cimetidine and (+)-tetrahydropalmatine ((+)-THP), significantly reduced NC accumulation and cytotoxicity in MDCK-hOCT2, MDCK-hOCT2/hMATE1 and rPCPT cells. Severe kidney damage with high levels of blood urea nitrogen and lactate dehydrogenase (LDH), reduced levels of alkaline phosphatase (ALP) and pathological changes were found in rats after 20days of successive i.v. doses of NC (5mg·kg(-1) ·day(-1) ). Concomitantly, the concentration of NC in the kidney reached similar high levels at 2h after the last dose of the 20 day treatment as those observed at 0.5 h after a single i.v. dose of 5mg·kg(-1) . Our data indicate that NC-induced nephrotoxicity might be mainly attributed to OCT2-mediated extensive renal uptake and weak tubular secretion by MATE1.

Identification of the High-affinity Substrate-binding Site of the Multidrug and Toxic Compound Extrusion (MATE) Family Transporter from Pseudomonas stutzeri

Multidrug and toxic compound extrusion (MATE) transporters exist in all three domains of life. They confer multidrug resistance by utilizing H(+) or Na(+) electrochemical gradients to extrude various drugs across the cell membranes. The substrate binding and the transport mechanism of MATE transporters is a fundamental process but so far not fully understood. Here we report a detailed substrate binding study of NorM_PS, a representative MATE transporter from Pseudomonas stutzeri Our results indicate that NorM_PS is a proton-dependent multidrug efflux transporter. Detailed binding studies between NorM_PS and 4',6-diamidino-2-phenylindole (DAPI) were performed by isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and spectrofluorometry. Two exothermic binding events were observed from ITC data, and the high-affinity event was directly correlated with the extrusion of DAPI. The affinities are about 1 μm and 0.1 mm for the high and low affinity binding, respectively. Based on our homology model of NorM_PS, variants with mutations of amino acids that are potentially involved in substrate binding, were constructed. By carrying out the functional characterization of these variants, the critical amino acid residues (Glu-257 and Asp-373) for high-affinity DAPI binding were determined. Taken together, our results suggest a new substrate-binding site for MATE transporters.

LCP crystallization and X-ray diffraction analysis of VcmN, a MATE transporter from Vibrio cholerae.

Multidrug and toxic compound extrusion (MATE) transporters, one of the multidrug exporter families, efflux xenobiotics towards the extracellular side of the membrane. Since MATE transporters expressed in bacterial pathogens contribute to multidrug resistance, they are important therapeutic targets. Here, a MATE-transporter homologue from Vibrio cholerae, VcmN, was overexpressed in Escherichia coli, purified and crystallized in lipidic cubic phase (LCP). X-ray diffraction data were collected to 2.5 Å resolution from a single crystal obtained in a sandwich plate. The crystal belonged to space group P212121, with unit-cell parameters a = 52.3, b = 93.7, c = 100.2 Å. As a result of further LCP crystallization trials, crystals of larger size were obtained using sitting-drop plates. X-ray diffraction data were collected to 2.2 Å resolution from a single crystal obtained in a sitting-drop plate. The crystal belonged to space group P212121, with unit-cell parameters a = 61.9, b = 91.8, c = 100.9 Å. The present work provides valuable insights into the atomic resolution structure determination of membrane transporters.