Multidrug And Toxin Extrusion Research Articles

MATEPRED-A-SVM-Based Prediction Method for Multidrug And Toxin Extrusion (MATE) Proteins

The growth and spread of drug resistance in bacteria have been well established in both mankind and beasts and thus is a serious public health concern. Due to the increasing problem of drug resistance, control of infectious diseases like diarrhea, pneumonia etc. is becoming more difficult. Hence, it is crucial to understand the underlying mechanism of drug resistance mechanism and devising novel solution to address this problem. Multidrug And Toxin Extrusion (MATE) proteins, first characterized as bacterial drug transporters, are present in almost all species. It plays a very important function in the secretion of cationic drugs across the cell membrane. In this work, we propose SVM based method for prediction of MATE proteins. The data set employed for training consists of 189 non-redundant protein sequences, that are further classified as positive (63 sequences) set comprising of sequences from MATE family, and negative (126 sequences) set having protein sequences from other transporters families proteins and random protein sequences taken from NCBI while in the test set, there are 120 protein sequences in all (8 in positive and 112 in negative set). The model was derived using Position Specific Scoring Matrix (PSSM) composition and achieved an overall accuracy 92.06%. The five-fold cross validation was used to optimize SVM parameter and select the best model. The prediction algorithm presented here is implemented as a freely available web server MATEPred, which will assist in rapid identification of MATE proteins.

Phosphorus and iron deficiencies induce a metabolic reprogramming and affect the exudation traits of the woody plant Fragaria×ananassa.

Strawberries are a very popular fruit among berries, for both their commercial and economic importance, but especially for their beneficial effects for human health. However, their bioactive compound content is strictly related to the nutritional status of the plant and might be affected if nutritional disorders (e.g. Fe or P shortage) occur. To overcome nutrient shortages, plants evolved different mechanisms, which often involve the release of root exudates. The biochemical and molecular mechanisms underlying root exudation and its regulation are as yet still poorly known, in particular in woody crop species. The aim of this work was therefore to characterize the pattern of root exudation of strawberry plants grown in either P or Fe deficiency, by investigating metabolomic changes of root tissues and the expression of genes putatively involved in exudate extrusion. Although P and Fe deficiencies differentially affected the total metabolism, some metabolites (e.g. raffinose and galactose) accumulated in roots similarly under both conditions. Moreover, P deficiency specifically affected the content of galactaric acid, malic acid, lysine, proline, and sorbitol-6-phosphate, whereas Fe deficiency specifically affected the content of sucrose, dehydroascorbic acid, galactonate, and ferulic acid. At the same time, the citrate content did not change in roots under both nutrient deficiencies with respect to the control. However, a strong release of citrate was observed, and it increased significantly with time, being +250% and +300% higher in Fe- and P-deficient plants, respectively, compared with the control. Moreover, concomitantly, a significant acidification of the growth medium was observed in both treatments. Gene expression analyses highlighted for the first time that at least two members of the multidrug and toxic compound extrusion (MATE) transporter family and one member of the plasma membrane H(+)-ATPase family are involved in the response to both P and Fe starvation in strawberry plants.

A subgroup of MATE transporter genes regulates hypocotyl cell elongation in Arabidopsis.

The growth of higher plants is under complex regulation to ensure the elaboration of developmental programmes under a changing environment. To dissect these regulatory circuits, we carried out genetic screens for Arabidopsis abnormal shoot (abs) mutants with altered shoot development. Here, we report the isolation of two dominant mutants, abs3-1D and abs4-1D, through activation tagging. Both mutants showed a 'bushy' loss of apical dominance phenotype. ABS3 and ABS4 code for two closely related putative Multidrug and Toxic Compound Extrusion (MATE) family of efflux transporters, respectively. ABS3 and ABS4, as well as two related MATE genes, ABS3-Like1 (ABS3L1) and ABS3L2, showed diverse tissue expression profiles but their gene products all localized to the late endosome/prevacuole (LE/PVC) compartment. The over-expression of these four genes individually led to the inhibition of hypocotyl cell elongation in the light. On the other hand, the quadruple knockout mutant (mateq) showed the opposite phenotype of an enhanced hypocotyl cell elongation in the light. Hypocotyl cell elongation and de-etiolation processes in the dark were also affected by the mutations of these genes. Exogenously applied sucrose attenuated the inhibition of hypocotyl elongation caused by abs3-1D and abs4-1D in the dark, and enhanced the hypocotyl elongation of mateq under prolonged dark treatment. We determined that ABS3 genetically interacts with the photoreceptor gene PHYTOCHROME B (PHYB). Our results demonstrate that ABS3 and related MATE family transporters are potential negative regulators of hypocotyl cell elongation and support a functional link between the endomembrane system, particularly the LE/PVC, and the regulation of plant cell elongation.

Translocation and accumulation of nicotine via distinct spatio-temporal regulation of nicotine transporters in Nicotiana tabacum

In plants, secondary metabolites play important roles in adaptation to the environment. Nicotine, a pyridine alkaloid in Nicotiana tabacum, functions as chemical barrier against herbivores. Nicotine produced in the root undergoes long-distance transport and accumulates mainly in the leaves. Since production of such defensive compounds is costly, plants must regulate the allocation of the products to their tissues; however, the molecular mechanism of nicotine translocation remains unclear. Our recent studies identified a novel multidrug and toxic compound extrusion (MATE)-type nicotine transporter, JAT2 (jasmonate-inducible alkaloid transporter 2). This transporter is specifically expressed in leaves, localizes to the tonoplast, and transports nicotine as its substrate. The specific induction of JAT2 expression in leaves by methyl jasmonate (MeJA) treatment suggests that this transporter plays an important role in nicotine distribution to leaves, especially under herbivore attack, by transporting nicotine into the vacuole. Considering JAT2, together with the previously identified MATE transporters JAT1, MATE1, and MATE2, and the PUP (purine permease) transporter NUP1 (nicotine uptake permease1), we show a model of nicotine translocation and accumulation via distinct spatio-temporal regulation of nicotine transporter expression. Furthermore, we discuss the possible role of nicotine transporters in determining outcrossing rates and seed production.

The importance of efflux systems in antibiotic resistance and efflux pump inhibitors in the management of resistance

The process of active efflux, the last described resistance mechanism in bacteria, is one of the important factors of acquired antibiotic resistance. Efflux systems which consist of membrane-located pump proteins that exist in all eukaryotic and prokaryotic cells, are responsible for extrusion of the various exogenous and endogenous substances. Bacterial pump proteins, namely ATP Binding Cassette (ABC), Major Facilitator (MFS), Small Multidrug Resistance (SMR), Multidrug and Toxic Compound Extrusion (MATE) and Resistance - Nodulation - Division (RND) are grouped in five super families. Efflux pumps play a role in intrinsic resistance against antibiotics among some pathogens. Besides, pumps give rise to acquired resistance by over-expression and contribute to other resistance mechanisms. Furthermore, pumps can directly or indirectly intensify the virulence properties of bacteria. Thus, for inhibition of efflux-mediated resistance or conversion of resistant bacteria to the susceptible phenotype, potential pump inhibitors combined with traditional antibiotics is one of the research areas of interest in the fight against resistance. There are some compounds known to inhibit bacterial pump systems, such as phenyl-arginine beta naphthylamide (PAβN), INF271, INF55, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), reserpine, 1-(1-naphthylmethyl)-piperazine (NMP), biricodar, timcodar, verapamil, milbemycin, chlorpromazine, paroxetine and omeprazole. However, particularly due to the toxicity problems, there has been no clinical use of a pump inhibitor yet. Nevertheless, in the upcoming period, derivatives of efflux pump inhibitor compounds with acceptable level of toxicity and efficacy may be included in antibacterial formulations. Additionally, a number of natural and synthetic compounds with pump inhibition activity are reported by many researchers in industrial and academic areas. In this context, various antibiotic derivatives (tetracycline, fluoroquinolone and aminoglycoside analogs) or non-antibiotic compounds (indole, urea, aromatic acid, piperidine-carboxylic acid, quinoline derivatives and peptidomimetics), some of which already have patent applications, are often studied. Pump inhibitors are shown to reduce the minimum inhibitory concentration (MIC) values of various antibiotics in some studies. In this review article, the structure and functions of bacterial efflux pumps have been summarized and the efflux pump inhibitors with natural and synthetic origins have been evaluated.

Isolation and characterization of differentially expressed genes in petals of chrysanthemum mutant cultivars developed by irradiation

Chrysanthemum is one of the most popular ornamental plants, whose petal colors are primarily determined by pigments including flavonoids/anthocyanins and carotenoids. To develop chrysanthemum cultivars with various petal colors, mutation breeding targeting alteration of pigmentation pattern has been performed. A radiation-induced mutant line, ‘ARTI-purple’, showed a flower color altered from the original bright pink to purple. In this study, we used suppression subtractive hybridization to analyze transcripts and characterize the differential gene expression of chrysanthemum petals between the mutant ‘ARTI-purple’ and its wild-type Chrysanthemum×morifolium cultivar ‘Argus’. One hundred and seventy-six genes were identified (e-value≤1e−5) and classified based on sequence homology to genes with known or putative functions. The genes were categorized functionally by gene ontology analysis and their tentative pathways were confirmed using the TAIR database. The analyses revealed that these genes were related to carbohydrate metabolism, biosynthesis of secondary metabolites, and lipid metabolism. Six genes in a Kyoto encyclopedia of genes and genomes (KEGG) pathway which included the largest number of differentially expressed genes were selected for validation by quantitative PCR, and most of them showed higher expression levels compared with the wild-type. In addition, we isolated two novel clones (PC06E06 and PC08C09) having glutathione S-transferase (GST) family conserved domains and one clone (PC02G08) having a Multidrug and toxic compound extrusion (MATE) family conserved domain based on analysis using conserved domain database (CDD). The expressions of PC08C09 and PC02G08 were upregulated in ‘ARTI-purple’, which implies that anthocyanin accumulation pattern might be altered in mutant. In this study, we identified several differentially expressed genes between ‘Argus’ and ‘ARTI-purple’. The analysis suggested that several metabolic genes as well as glutathione S-transferases and MATEs might be involved in the control of flower pigmentation in chrysanthemum.

Structure of an H+‐coupled, Substrate‐bound MATE Transporter Yields Mechanistic Insights into Multidrug Extrusion

Multidrug and toxic compound extrusion (MATE) proteins constitute a ubiquitous family of multidrug transporters and couple the efflux of structurally dissimilar drugs to the influx of either Na+ or H+. The ~900 MATE transporters identified thus far can be classified into the NorM, DinF (DNA‐damage‐inducible protein F) and eukaryotic subfamilies based on amino‐acid sequence similarity. Structures of Na+‐coupled, extracellular‐facing NorM transporters had been determined, which revealed twelve membrane‐spanning segments that are related by a quasi‐twofold rotational symmetry as well as a multidrug‐binding cavity situated near the membrane‐periplasm interface. Here we report the crystal structures of an H+‐coupled MATE transporter from the DinF subfamily, with and without substrate, unveiling a surprisingly asymmetric arrangement of twelve transmembrane helices and a largely hydrophobic multidrug‐binding chamber located in the middle of the lipid bilayer. Combining structural and biochemical analyses, we confirmed the biological relevance of the substrate‐binding site and suggested a direct competition between H+ and substrate during DinF‐mediated drug transport. Our findings provided fundamental brushstrokes to the molecular picture depicting how a MATE transporter works and laid the groundwork for future experimental efforts aimed at overcoming multidrug resistance.

Identification and expression analysis of MATE genes involved in flavonoid transport in blueberry plants.

Multidrug and toxic compound extrusion (MATE) proteins are the most recently identified family of multidrug transporters. In plants, this family is remarkably large compared to the human and bacteria counterpart, highlighting the importance of MATE proteins in this kingdom. Here 33 Unigenes annotated as MATE transporters were found in the blueberry fruit transcriptome, of which eight full-length cDNA sequences were identified and cloned. These proteins are composed of 477–517 residues, with molecular masses ~54 kDa, and theoretical isoelectric points from 5.35 to 8.41. Bioinformatics analysis predicted 10–12 putative transmembrane segments for VcMATEs, and localization to the plasma membrane without an N-terminal signal peptide. All blueberry MATE proteins shared 32.1–84.4% identity, among which VcMATE2, VcMATE3, VcMATE5, VcMATE7, VcMATE8, and VcMATE9 were more similar to the MATE-type flavonoid transporters. Phylogenetic analysis showed VcMATE2, VcMATE3, VcMATE5, VcMATE7, VcMATE8 and VcMATE9 clustered with MATE-type flavonoid transporters, indicating that they might be involved in flavonoid transport. VcMATE1 and VcMATE4 may be involved in the transport of secondary metabolites, the detoxification of xenobiotics, or the export of toxic cations. Real-time quantitative PCR demonstrated that the expression profile of the eight VcMATE genes varied spatially and temporally. Analysis of expression and anthocyanin accumulation indicated that there were some correlation between the expression profile and the accumulation of anthocyanins. These results showed VcMATEs might be involved in diverse physiological functions, and anthocyanins across the membranes might be mutually maintained by MATE-type flavonoid transporters and other mechanisms. This study will enrich the MATE-based transport mechanisms of secondary metabolite, and provide a new biotechonology strategy to develop better nutritional blueberry cultivars.

A transporter for abiotic stress and plant metabolite resistance in the ectomycorrhizal fungus Tricholoma vaccinum.

Fungi exposed to toxic substances including heavy metals, xenobiotics, or secondary metabolites formed by co-occurring plants or other microorganisms require a detoxification system provided by exporters of several classes of transmembrane proteins. In case of mycorrhiza, plant metabolites need to be exported at the plant interface, while the extraradical hyphae may prevent heavy metal uptake, thus acting as a biofilter to the host plant at high environmental concentrations. One major family of such transporter proteins is the multidrug and toxic compound extrusion (MATE) class, a member of which, Mte1, was studied in the ectomycorrhizal fungus Tricholoma vaccinum. Phylogenetic analyses placed the protein in a subgroup of basidiomycete MATE sequences. The gene mte1 was found to be induced during symbiotic interaction. It mediated detoxification of xenobiotics and metal ions such as Cu, Li, Al, and Ni, as well as secondary plant metabolites if heterologously expressed in Saccharomyces cerevisiae.

YeeO from Escherichia coli exports flavins

Multidrug and toxic compound extrusion (MATE) proteins help maintain cellular homeostasis by secreting metabolic wastes. Flavins may occur as cellular waste products, with their production and secretion providing potential benefit for industrial applications related to biofuel cells. Here we find that MATE protein YeeO from Escherichia coli exports both flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Significant amounts of flavins were trapped intracellularly when YeeO was produced indicating transport limits secretion of flavins. Wild-type E. coli secreted 3 flavins (riboflavin, FMN, and FAD), so E. coli likely produces additional flavin transporters.

Involvement of the leaf-specific multidrug and toxic compound extrusion (MATE) transporter Nt-JAT2 in vacuolar sequestration of nicotine in Nicotiana tabacum.

Alkaloids play a key role in higher plant defense against pathogens and herbivores. Following its biosynthesis in root tissues, nicotine, the major alkaloid of Nicotiana species, is translocated via xylem transport toward the accumulation sites, leaf vacuoles. Our transcriptome analysis of methyl jasmonate-treated tobacco BY-2 cells identified several multidrug and toxic compound extrusion (MATE) transporter genes. In this study, we characterized a MATE gene, Nicotiana tabacum jasmonate-inducible alkaloid transporter 2 (Nt-JAT2), which encodes a protein that has 32% amino acid identity with Nt-JAT1. Nt-JAT2 mRNA is expressed at a very low steady state level in whole plants, but is rapidly upregulated by methyl jasmonate treatment in a leaf-specific manner. To characterize the function of Nt-JAT2, yeast cells were used as the host organism in a cellular transport assay. Nt-JAT2 was localized at the plasma membrane in yeast cells. When incubated in nicotine-containing medium, the nicotine content in Nt-JAT2-expressing cells was significantly lower than in control yeast. Nt-JAT2-expressing cells also showed lower content of other alkaloids like anabasine and anatabine, but not of flavonoids, suggesting that Nt-JAT2 transports various alkaloids including nicotine. Fluorescence assays in BY-2 cells showed that Nt-JAT2-GFP was localized to the tonoplast. These findings indicate that Nt-JAT2 is involved in nicotine sequestration in leaf vacuoles following the translocation of nicotine from root tissues.

Abstract 4639: Do cation-selective transporters help or hurt the antitumor efficacy of metformin in breast cancer

Abstract Introduction Metformin is effective against many cancers including breast cancer. Activation of intracellular adenosine monophosphate-activated protein kinase (AMPK) is implied in metformin anticancer efficacy. Due to its hydrophilicity and positive charge, metformin relies on cation-selective transporters for cellular uptake. Accordingly, metformin cellular uptake, AMPK activation and anti-proliferative effects increased when organic cation transporter 3 (OCT3) was expressed in BT20 cells (BT20-OCT3) that have very low levels of cation-selective transporters (Cai et al., AAPS Abstract W4368, 2013). Our previous studies showed that human breast cancer cell lines and tumors exhibit wide variability in expression of metformin transporters, e.g. OCT1-3, plasma monoamine transporter (PMAT), and multidrug and toxin extrusion (MATE) transporters 1-2 (Zhang et al., AACR Abstract 9800, 2012). This study aims to assess the pivotal role of transporters in metformin antitumor effects using xenograft mice with tumors from BT20 and OCT3-BT20 cells. Methods BT20 cells stably transfected with OCT3 were produced, and OCT3 activity verified by [14C]metformin (50µM) uptake in presence/absence of 50µM famotidine (OCT3 and MATE1 inhibitor) or 500 µM quinidine (pan transporter inhibitor). Optimum metformin dose for tumor study was assessed by a dose-range pharmacokinetic study in mice dosed intraperitoneally (IP) with 15, 30, 50, 100, and 150mg/kg [14C]metformin. Xenograft mice were produced using 2×106 BT20/OCT3-BT20 cells. Doxorubicin (DOX; 4mg/kg) or DOX + 100mg/kg metformin was given IP every 5 days, and tumor volumes measured over 25 days. Results Metformin plasma AUC0-24hr was linear up to 100 mg/kg (AUC0-24hr of 95, 160, 366, 770 and 510 µmol*hr/L at 15, 30, 50, 100 and 150 mg/kg, respectively); hence, this dose was used for antitumor efficacy study. Surprisingly, tumors were larger in DOX + metformin group compared to DOX (45.3 vs. 23.5 mm3) in mice with OCT3-BT20 tumors. In mice with BT20 tumors, tumors were smaller in DOX + metformin group compared to DOX (20.5 vs. 61.8 mm3). Since BT20 tumors grew slowly, tumor measurements were possible in only 2 mice/group. Hence, the more aggressive MCF-7 human breast cancer cell was used to develop an OCT3-expressing (OCT3-MCF7) clone in which metformin uptake was 4.5-fold higher than in MCF-7 cells (65.66 vs. 15.17 pmol/mg protein/min). Metformin antitumor effect in xenograft mice with MCF-7 and OCT3-MCF7 tumors is under evaluation. Conclusion In vitro data imply that OCT3 expression in human breast cancer cells enhances metformin cellular uptake and anti-proliferative effect. In vivo xenograft mice studies show that BT20 tumors over-expressing OCT3 are less responsive to metformin + DOX than native BT20 tumors; however, further studies are needed to show statistical significance. Citation Format: Hao Cai, Muhammad Wahajuddin, Ruth Everett, Dhiren R. Thakker. Do cation-selective transporters help or hurt the antitumor efficacy of metformin in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4639. doi:10.1158/1538-7445.AM2014-4639

X-ray crystallographic analysis of a MATE multidrug transporter from V. cholerae

MATE (Multidrug And Toxic compound Extrusion) family transporters are highly conserved from Bacteria to Eukarya including human, and export a broad range of xenobiotics using either a proton or a sodium ion gradient across the membrane. Especially in bacterial pathogens, MATE transporters contribute to their multiple drug resistance (MDR). To understand how MATE transporters export various substrates such as drugs and thus how pathogens acquire MDR, structural analyses are essential. The crystal structures of several MATE transporters from pathogens have been reported. However, because of the limited resolution and the lack of drug-MATE transporters complex structures, the recognition mechanism of various substrates and the coupling mechanism of the cation influx and the drug efflux have been poorly understood. Although the high-resolution structures of MATE transporters from non-pathogenic archaeal P. furiosus (PfMATE) have been reported, PfMATE shares low sequence identity with MATE transporters from pathogens such as V. cholerae. Therefore, further findings of the structural mechanism of MDR caused by MATE transporters from pathogens have been needed. To understand the substrate recognition and transport mechanism of MATE transporters from pathogens, we determined the crystal structures of one of MATE transporters from V. cholerae (VcMATE) at 2.5-2.7 Å resolutions using in meso crystallization method. The high-resolution structures of VcMATE show two distinct conformations, as observed in the structures of PfMATE, and reveal the large movement of transmembrane helix 1 and the putative substrate-binding site. The structures suggest that the bending of transmembrane helix 1 and the sequential collapse of the putative substrate-binding site induce the release of the bound substrate. This conformational change during the substrate transport may be a common mechanism among MATE transporters from pathogens to non-pathogens.

Beyond cellular detoxification: a plethora of physiological roles for MDR transporter homologs in plants.

Higher plants possess a multitude of Multiple Drug Resistance (MDR) transporter homologs that group into three distinct and ubiquitous families—the ATP-Binding Cassette (ABC) superfamily, the Major Facilitator Superfamily (MFS), and the Multidrug And Toxic compound Extrusion (MATE) family. As in other organisms, such as fungi, mammals, and bacteria, MDR transporters make a primary contribution to cellular detoxification processes in plants, mainly through the extrusion of toxic compounds from the cell or their sequestration in the central vacuole. This review aims at summarizing the currently available information on the in vivo roles of MDR transporters in plant systems. Taken together, these data clearly indicate that the biological functions of ABC, MFS, and MATE carriers are not restricted to xenobiotic and metal detoxification. Importantly, the activity of plant MDR transporters also mediates biotic stress resistance and is instrumental in numerous physiological processes essential for optimal plant growth and development, including the regulation of ion homeostasis and polar transport of the phytohormone auxin.

Brassica oleracea MATE encodes a citrate transporter and enhances aluminum tolerance in Arabidopsis thaliana.

The secretion of organic acid anions from roots is an important mechanism for plant aluminum (Al) tolerance. Here we report cloning and characterizing BoMATE (KF031944), a multidrug and toxic compound extrusion (MATE) family gene from cabbage (Brassica oleracea). The expression of BoMATE was more abundant in roots than in shoots, and it was highly induced by Al treatment. The (14)C-citrate efflux experiments in oocytes demonstrated that BoMATE is a citrate transporter. Electrophysiological analysis and SIET analysis of Xenopus oocytes expressing BoMATE indicated BoMATE is activated by Al. Transient expression of BoMATE in onion epidermal cells demonstrated that it localized to the plasma membrane. Compared with the wild-type Arabidopsis, the transgenic lines constitutively overexpressing BoMATE enhanced Al tolerance and increased citrate secretion. In addition, Arabidopsis transgenic lines had a lower K(+) efflux and higher H(+) efflux, in the presence of Al, than control wild type in the distal elongation zone (DEZ). This is the first direct evidence that MATE protein is involved in the K(+) and H(+) flux in response to Al treatment. Taken together, our results show that BoMATE is an Al-induced citrate transporter and enhances aluminum tolerance in Arabidopsis thaliana.

The contribution of human OCT1, OCT3, and CYP3A4 to nitidine chloride-induced hepatocellular toxicity.

Nitidine chloride (NC), a quaternary ammonium alkaloid, has numerous pharmacological effects, such as anticancer activity. However, it was found that NC also has hepatocellular toxicity. Because organic cation transporters 1 and 3 (OCT1 and OCT3) might mediate the influx of NC into hepatocytes, multidrug and toxin extrusion 1 (MATE1) probably mediates the efflux of NC from hepatocytes, while cytochrome P450 (P450) enzymes might contribute to NC metabolism, the present study was to evaluate the contribution of OCT1, OCT3, MATE1, and P450 enzymes to NC-induced hepatocellular toxicity. Our results showed that the uptake of NC in Madin-Darby canine kidney (MDCK) cells expressing human (h) OCT1 and OCT3 (MDCK-hOCT1 and MDCK-hOCT3) was significantly higher than that in mock cells; the hOCT1- and hOCT3-mediated uptake followed typical Michaelis-Menten kinetics. Meanwhile, NC was also a substrate of hMATE1, although its transport capacity was much lower than that of OCT1 NC-induced cytotoxicity in MDCK-hOCT1 or MDCK-hOCT3 cells was obviously higher than that in mock cells. Quinidine and (+)-tetrahydropalmatine [(+)-THP], OCT1 and OCT3 inhibitors, significantly reduced the uptake of NC in MDCK-hOCT1 cells, MDCK-hOCT3 cells, and rat primary hepatocytes, but only (+)-THP markedly attenuated the NC-induced toxicity. In addition, P450 enzymes, such as CYP3A4, mediated the metabolism of NC, and NC-induced toxicity in MDCK-hOCT1/hCYP3A4 cells was lower than that in MDCK-hOCT1 cells. Our results indicated that NC is a substrate of hOCT1, hOCT3, and CYP3A4; that OCT1 and OCT3 mediate the uptake of NC in hepatocytes and subsequently cause hepatotoxicity; and that NC-induced toxicity could be attenuated by CYP3A4-mediated metabolism.

VvMATE1 and VvMATE2 encode putative proanthocyanidin transporters expressed during berry development in Vitis vinifera L.

VvMATE1 and VvMATE2 encode putative PA transporters expressed during seed development in grapevine. The subcellular localization of these MATE proteins suggests different routes for the intracellular transport of PAs. Proanthocyanidins (PAs), also called condensed tannins, protect plants against herbivores and are important quality components of many fruits. PAs biosynthesis is part of the flavonoid pathway that also produces anthocyanins and flavonols. In grape fruits, PAs are present in seeds and skin tissues. PAs are synthesized in the cytoplasm and accumulated into the vacuole and apoplast; however, little is known about the mechanisms involved in the transport of these compounds to such cellular compartments. A gene encoding a Multidrug And Toxic compound Extrusion (MATE) family protein suggested to transport anthocyanins-named VvMATE1-was used to identify a second gene of the MATE family, VvMATE2. Analysis of their deduced amino acid sequences and the phylogenetic relationship with other MATE-like proteins indicated that VvMATE1 and VvMATE2 encode putative PA transporters. Subcellular localization assays in Arabidopsis protoplasts transformed with VvMATE-GFP fusion constructs along with organelle-specific markers revealed that VvMATE1 is localized in the tonoplast whereas VvMATE2 is localized in the Golgi complex. Major expression of both genes occurs during the early stages of seed development concomitant with the accumulation of PAs. Both genes are poorly expressed in the skin of berries while VvMATE2 is also expressed in leaves. The presence of putative cis-acting elements in the promoters of VvMATE1 and VvMATE2 may explain the differential transcriptional regulation of these genes in grapevine. Altogether, these results suggest that these MATE proteins could mediate the transport and accumulation of PAs in grapevine through different routes and cellular compartments.

Molecular mechanisms of Al tolerance in gramineous plants

Aluminum (Al) toxicity has limited the productivity and expansion of cereal crops on acid soils; however, a number of plant species or cultivars have developed different strategies for detoxifying aluminum both internally and externally. This review focuses on recent progress on molecular mechanisms of Al tolerance in gramineous plants. A common mechanism in all gramineous plants is the secretion of organic acid anions (citrate and malate) from the roots. Genes belonging to ALMT (for Aluminum-activated malate transporter) and MATE (Multidrug and toxic compound extrusion) family involved in the secretion have been identified in several plant species; however, different plant species show different gene expression patterns including Al-induction, spatial and temporal expression, and tissue localization. Furthermore, the mechanisms regulating the gene expression also differ with plant species, which are achieved by increased tandem repeated element, increase of copy number, insertion of transposon, or alteration of cis-acting element. In addition to these common Al exclusion mechanisms, rice as a highly Al-tolerant species has developed a number of other mechanisms for detoxification of Al. A transcription factor for Al tolerance ART1 identified in rice regulates at least 30 genes implicated in internal and external detoxification of Al. These multiple genes may contribute to the high Al tolerance of rice. In the future, regulation mechanisms of Al-tolerance genes need to be further investigated.

ADP1 Affects Plant Architecture by Regulating Local Auxin Biosynthesis

Plant architecture is one of the key factors that affect plant survival and productivity. Plant body structure is established through the iterative initiation and outgrowth of lateral organs, which are derived from the shoot apical meristem and root apical meristem, after embryogenesis. Here we report that ADP1, a putative MATE (multidrug and toxic compound extrusion) transporter, plays an essential role in regulating lateral organ outgrowth, and thus in maintaining normal architecture of Arabidopsis. Elevated expression levels of ADP1 resulted in accelerated plant growth rate, and increased the numbers of axillary branches and flowers. Our molecular and genetic evidence demonstrated that the phenotypes of plants over-expressing ADP1 were caused by reduction of local auxin levels in the meristematic regions. We further discovered that this reduction was probably due to decreased levels of auxin biosynthesis in the local meristematic regions based on the measured reduction in IAA levels and the gene expression data. Simultaneous inactivation of ADP1 and its three closest homologs led to growth retardation, relative reduction of lateral organ number and slightly elevated auxin level. Our results indicated that ADP1-mediated regulation of the local auxin level in meristematic regions is an essential determinant for plant architecture maintenance by restraining the outgrowth of lateral organs.

Drug Extrusion Process of Mate Multidrug Efflux Transporter Revealed by Molecular Dynamics Simulations

MATE (the Multidrug And Toxic compound Extrusion) is one of the five multidrug efflux transporter super-families. It is featured as primary secondary active transporter and comprises of 12 trans-membrane helices. The first and last six transmembrane helices (TMs) form N-lobe and C-lobe domains, showing a two-fold rotational symmetry. It is considered that the drug release/capture related motions are characterized by switching of inward/outward-facing conformations (rocker-switch model).Recently, multiple structures of the MATE from P.furiosus (PfMATE), has been determined by X-ray crystallography in outward-facing conformation. Two distinct structures ‘Straight’ and ‘Bent’ are characterized by conformations of TM1. Furthermore, PfMATE is shown to be H+ driven antiporter, suggesting that D41 and D184 in N-lobe are involved as important proton pathway. It is also interesting that MATE has the hydrophobic surface of inner cavity, since other multidrug transporters exhibit hydrophilic. The sharply opened interface to the lipid molecules suggests the possibilities that the lipid molecules intensively interact with the inside of the cavity.To further elucidate the drug release mechanism in outward facing stage, we adopted several promising simulation methods, continuum electrostatic analysis to predict protonation states, a number of independent all-atom Molecular Dynamics (MD) simulations by changing conditions (initial conformations, protonations, lipid positions, etc.) for dynamics and interactions, and quantum mechanics calculation for drug forcefield development. Our results suggest that D41 is protonated in Straight, while D41 and D184 are both protonated in Bent. Furthermore, we successfully observed multiple drug release events in some conditions of MD simulations. In the poster presentation, we will report and discuss the dynamics and interactions of PfMATE revealed by different conditions of MDs. We will also discuss the insights of the drug release mechanism of PfMATE obtained by the simulations.