Multidrug And Toxic Compound Extrusion Transporters Research Articles

Genome-wide identification of MATE and ALMT genes and their expression profiling in mungbean (Vigna radiata L.) under aluminium stress

The Multidrug and toxic compound extrusion (MATE) and aluminium activated malate transporter (ALMT) gene families are involved in response to aluminium (Al) stress. In this study, we identified 48 MATE and 14 ALMT gene families in Vigna radiata genome and classified into 5 (MATE) and 3 (ALMT) clades by phylogenetic analysis. All the VrMATE and VrALMT genes were distributed across mungbean chromosomes. Tandem duplication was the main driving force for evolution and expansion of MATE gene family. Collinearity of mungbean with soybean indicated that MATE gene family is closely linked to Glycine max. Eight MATE transporters in clade 2 were found to be associated with previously characterized Al tolerance related MATEs in various plant species. Citrate exuding motif (CEM) was present in seven VrMATEs of clade 2. Promoter analysis revealed abundant plant hormone and stress responsive cis-elements. Results from quantitative real time-polymerase chain reaction (qRT-PCR) revealed that VrMATE19, VrMATE30 and VrALMT13 genes were markedly up-regulated at different time points under Al stress. Overall, this study offers a new direction for further molecular characterization of the MATE and ALMT genes in mungbean for Al tolerance.

Genome-wide mining and characterization of MATE transporters in Coriandrum sativum L.

Multidrug and Toxic Compound Extrusion (MATE) proteins are responsible for the transport of a wide range of metabolites out of plant cells. This helps to protect the cells from toxins and other harmful compounds. MATE proteins also play a role in plant development, by regulating the transport of hormones and other signalling molecules. They transport a wide variety of substances, including organic acids, plant hormones, flavonoids, alkaloids, terpenes and other secondary metabolites. MATE proteins are thought to play similar roles in Coriander, in addition to stress responses. The MATE genes in the coriander genome have been identified and characterized. Detailed genome homology search and domain identification analysis have identified 91 MATE proteins in the genome assembly of coriander. A phylogenetic analysis of the identified proteins divided them into five major clades. The functions of the transporters in each cluster were predicted based on the clustering pattern of the functionally characterized proteins. The amino acid sequences, exon-intron structures and motif details of all the 91 proteins are identified and described. This is the first work on the MATE transporters in coriander and the results deliver clues for the molecular mechanisms behind the stress responses and secondary metabolite transport in coriander.

Genome-wide identification of MATE, functional analysis and molecular dynamics of DcMATE21 involved in anthocyanin accumulation in Daucus carota

Anthocyanins are a subclass of flavonoids that are synthesized in the endoplasmic reticulum and then transported to the vacuole in plants. Multidrug and toxic compound extrusion transporters (MATE) is a family of membrane transporters that transport ions and secondary metabolites, such as anthocyanins, in plants. Although various studies on MATE transporters have been carried out on different plant species, this is the first comprehensive report to mine the Daucus carota genome to identify the MATE gene family. Our study identified 45 DcMATEs through genome-wide analysis and detected five segmental and six tandem duplications from the genome. The chromosome distribution, phylogenetic analysis, and cis-regulatory elements revealed the structural diversity and numerous functions associated with the DcMATEs. In addition, we analyzed RNA-seq data obtained from the European Nucleotide Archive to screen for the expression of DcMATEs involved in anthocyanin biosynthesis. Among the identified DcMATEs, DcMATE21 correlated with anthocyanin content in the different D. carota varieties. In addition, the expression of DcMATE21 and anthocyanin biosynthesis genes was correlated under abscisic acid, methyl jasmonate, sodium nitroprusside, salicylic acid, and phenylalanine treatments, which were substantiated by anthocyanin accumulation in the in vitro cultures. Further molecular membrane dynamics of DcMATE21 with anthocyanin (cyanidin-3-glucoside) identified the binding pocket, showing extensive H-bond interactions with 10 crucial amino acids present in the transmembrane helix of 7, 8, and 10 of DcMATE21. The current investigation, using RNA-seq, in vitro cultures, and molecular dynamics studies revealed the involvement of DcMATE21 in anthocyanin accumulation in vitro cultures of D. carota.

Genome wide identification and characterization of MATE family genes in mangrove plants.

Multidrug and Toxic Compound Extrusion (MATE) proteins are essential transporters that extrude metabolites and participate in plant development and cellular detoxification. MATE transporters, which play crucial roles in the survival of mangrove plants under highly challenged environments, by specialized salt extrusion mechanisms, are mined from their genomes and reported here for the first time. Through homology search and domain prediction in the genome assemblies of Avicennia marina, Bruguiera sexangula, Ceriops zippeliana, Kandelia obovata, Rhizophora apiculata and Ceriops tagal, 74, 68, 66, 66, 63 and 64 MATE proteins, respectively were identified. The phylogenetic analysis divided the identified proteins into five major clusters and following the clustering pattern of the functionally characterized proteins, functions of the transporters in each cluster were predicted. Amino acid sequences, exon-intron structure, motif details and subcellular localization pattern for all the 401 proteins are described. The custom designed repeat masking libraries generated for each of these genomes, which will be of extensive use for the researchers worldwide, are also provided in this paper. This is the first study on the MATE genes in mangroves and the results provide comprehensive information on the molecular mechanisms enabling the survival of mangroves under hostile conditions.

A single nucleotide substitution in the MATE transporter gene regulates plastochron and the many noded dwarf phenotype in barley (Hordeum vulgare L.)

In higher plants, the shoot apical meristem produces lateral organs in a regular spacing (phyllotaxy) and timing (plastochron). The molecular analysis of mutants associated with phyllotaxy and plastochron would increase our understanding of the mechanism of shoot architecture formation. In this study, we identified mutant mnd8ynp5 that shows an increased rate of leaf emergence and a larger number of nodes in combination with a dwarfed growth habit from an EMS-treated population of the elite barley cultivar Yangnongpi 5. Using a map-based cloning strategy, the mnd8 gene was narrowed down to a 6.7-kb genomic interval on the long arm of chromosome 5H. Sequence analysis revealed that a C to T single-nucleotide mutation occurred at the first exon (position 953) of HORVU5Hr1G118820, leading to an alanine (Ala) to valine (Val) substitution at the 318th amino acid site. Next, HORVU5Hr1G118820 was defined as the candidate gene of MND8 encoding 514 amino acids and containing two multidrug and toxic compound extrusion (MATE) domains. It is highly homologous to maize Bige1 and has a conserved function in the regulation of plant development by controlling the leaf initiation rate. Examination of modern barely varieties showed that Hap-1 was the dominant haplotype and was selected in barley breeding around the world. Collectively, our results indicated that mnd8ynp5 is a novel allele of the HORVU5Hr1G118820 gene that is possibly responsible for the shortened plastochron and many noded dwarf phenotype in barley.

Genome-wide identification and expression analysis of MATE gene family in citrus fruit (Citrus clementina)

Multidrug and toxic compound extrusion (MATE) proteins are a class of secondary active multidrug transporters. In plants, this family has significantly expanded and is involved in numerous plant physiological processes. Although MATE proteins have been identified in an increasing number of species, the understanding about this family in citrus remains unclear. In this study, a total of 69 MATE transporters were identified in the citrus genome (Citrus clementina) and classified into four groups by phylogenetic analysis. Tandem and segmental duplication events were the main causes of the citrus MATE family expansion. RNA-seq and qRT-PCR analyses were performed during citrus fruit development. The results indicated that CitMATE genes showed specific expression profiles in citrus peels and flesh at different developmental stages. Combined with the variations of flavonoids and citrate levels in citrus fruit, we suggested that CitMATE43 and CitMATE66 may be involved in the transport process of flavonoids and citrate in citrus fruit, respectively. In addition, two flavonoids positive regulators, CitERF32 and CitERF33, both directly bind to and activated the CitMATE43 promoter. Our results provide comprehensive information on citrus MATE genes and valuable understanding for the flavonoids and citrate metabolism in citrus fruit.

Genome-Wide Analysis of Multidrug and Toxic Compound Extruction Transporters in Grape.

Grape (Vitis vinifera L.) is an important fruit crop in the world. It is used as a table grape and is also used for raisin and wine production. Grape berries accumulate secondary metabolites, such as anthocyanins, tannins, and resveratrol, which are known as functional compounds for human health. Multidrug and toxic compound extrusion transporter (MATEs) transport secondary metabolites. MATEs also transport other solutes, including organic acids, and toxic xenobiotics, depending on cation gradient and play various roles in plants. MATE comprises 300–500 amino acid residues and possesses a MATE domain and 8–12 transmembrane domains. In the present study, 59 MATE genes were identified in the grape genome, and phylogenetic analysis revealed the presence of four groups of grape MATEs (Group 1–4). Their information, such as gene structures, protein motifs, predicted subcellular localizations, and gene IDs of four genome annotations, that is, CRIBI v1, CRIBI v2, Genoscope, and Vcost v3, were annotated. The transport substrates and physiological functions of grape MATEs were estimated based on their homology with the analyzed MATEs in other plant species. Group 1 may transport toxic compounds and alkaloids, Group 2 may transport polyphenolic compounds, Group 3 may transport organic acids, and Group 4 may transport plant hormones related to signal transduction. In addition to the known anthocyanin transporters, VvMATE37 and VvMATE39, a novel anthocyanin transporter, VvMATE38 in Group 2, was suggested as a key transporter for anthocyanin accumulation in grape berry skin. VvMATE46, VvMATE47, and VvMATE49 in Group 3 may contribute to Al3+ detoxification and Fe2+/Fe3+ translocation via organic acid transport. This study provides helpful and fundamental information for grape MATE studies and resolves the confusion of gene IDs in different genome annotations.

Two homeologous MATE transporter genes, NtMATE21 and NtMATE22, are involved in the modulation of plant growth and flavonol transport in Nicotiana tabacum.

The multidrug and toxic compound extrusion (MATE) protein family has been implicated in the transport of a diverse range of molecules, including specialized metabolites. In tobacco (Nicotiana tabacum), only a limited number of MATE transporters have been functionally characterized, and no MATE transporter has been studied in the context of flavonoid transport in this plant species so far. In the present study, we characterize two homeologous tobacco MATE genes, NtMATE21 and NtMATE22, and demonstrate their role in flavonol transport and in plant growth and development. The expression of these two genes was reported to be up-regulated in trichomes as compared with the trichome-free leaf. The transcript levels of NtMATE21 and NtMATE22 were found to be higher in flavonol overproducing tobacco transgenic lines as compared with wild type tobacco. The two transporters were demonstrated to be localized to the plasma membrane. Genetic manipulation of NtMATE21 and NtMATE22 led to altered growth phenotypes and modulated flavonol contents in N. tabacum. The β-glucuronidase and green fluorescent protein fusion transgenic lines of promoter regions suggested that NtMATE21 and NtMATE22 are exclusively expressed in the trichome heads in the leaf tissue and petals. Moreover, in a transient transactivation assay, NtMYB12, a flavonol-specific MYB transcription factor, was found to transactivate the expression of NtMATE21 and NtMATE22 genes. Together, our results strongly suggest the involvement of NtMATE21 and NtMATE22 in flavonol transport as well as in the regulation of plant growth and development.

The Roles of Multidrug and Toxic Compound Extrusion (MATE) Transporters in Regulating Agronomic Traits

Multidrug and toxic compound extrusion (MATE) transporters are ancient proteins conserved among various kingdoms, from prokaryotes to eukaryotes. In plants, MATEs usually form a large family in the genome. Homologous MATE transporters have different subcellular localizations, substrate specificities, and responses to external stimuli for functional differentiations. The substrates of MATEs in plants include polyphenols, alkaloids, phytohormones, and ion chelators. The accumulation of these substrates is often associated with favorable agronomic traits such as seed and fruit colors, the balance between dormancy and germination, taste, and stress adaptability. In crops, wild germplasms and domesticated germplasms usually have contrasting agronomic traits such as seed color, seed taste, and stress tolerance. MATE transporters are involved in the regulations of these traits. In this review, we discuss the uniqueness and significance of there being such a large family of MATEs in plants, their substrate diversity that enables them to be involved in various agronomic traits, and the allelic forms and the expression patterns of MATE that are associated with favorable agronomic traits in domesticated crops. The understanding on the roles of MATEs in regulating favorable agronomic traits in crops will provide hints for the selection of genes for molecular breeding that improve desirable traits.

The Tiny Companion Matters: The Important Role of Protons in Active Transports in Plants.

In plants, the translocation of molecules, such as ions, metabolites, and hormones, between different subcellular compartments or different cells is achieved by transmembrane transporters, which play important roles in growth, development, and adaptation to the environment. To facilitate transport in a specific direction, active transporters that can translocate their substrates against the concentration gradient are needed. Examples of major active transporters in plants include ATP-binding cassette (ABC) transporters, multidrug and toxic compound extrusion (MATE) transporters, monosaccharide transporters (MSTs), sucrose transporters (SUTs), and amino acid transporters. Transport via ABC transporters is driven by ATP. The electrochemical gradient across the membrane energizes these secondary transporters. The pH in each cell and subcellular compartment is tightly regulated and yet highly dynamic, especially when under stress. Here, the effects of cellular and subcellular pH on the activities of ABC transporters, MATE transporters, MSTs, SUTs, and amino acid transporters will be discussed to enhance our understanding of their mechanics. The relation of the altered transporter activities to various biological processes of plants will also be addressed. Although most molecular transport research has focused on the substrate, the role of protons, the tiny counterparts of the substrate, should also not be ignored.

Identifying Quantitative Trait Loci and Candidate Genes Conferring Resistance to Soybean Mosaic Virus SC7 by Quantitative Trait Loci-Sequencing in Soybean.

Soybean mosaic virus (SMV) is detrimental to soybean (Glycine max) breeding, seed quality, and yield worldwide. Improving the basic resistance of host plants is the most effective and economical method to reduce damage from SMV. Therefore, it is necessary to identify and clone novel SMV resistance genes. Here, we report the characterization of two soybean cultivars, DN50 and XQD, with different levels of resistance to SMV. Compared with XQD, DN50 exhibits enhanced resistance to the SMV strain SC7. By combining bulked-segregant analysis (BSA)-seq and fine-mapping, we identified a novel resistance locus, RSMV-11, spanning an approximately 207-kb region on chromosome 11 and containing 25 annotated genes in the reference Williams 82 genome. Of these genes, we identified eleven with non-synonymous single-nucleotide polymorphisms (SNPs) or insertion-deletion mutations (InDels) in their coding regions between two parents. One gene, GmMATE68 (Glyma.11G028900), harbored a frameshift mutation. GmMATE68 encodes a multidrug and toxic compound extrusion (MATE) transporter that is expressed in all soybean tissues and is induced by SC7. Given that MATE transporter families have been reported to be linked with plant disease resistance, we suggest that GmMATE68 is responsible for SC7 resistance in DN50. Our results reveal a novel SMV-resistance locus, improving understanding of the genetics of soybean disease resistance and providing a potential new tool for marker-assisted selection breeding in soybean.

The Poly-Glutamate Motif of GmMATE4 Regulates Its Isoflavone Transport Activity.

Multidrug and toxic compound extrusion (MATE) transporters in eukaryotes have been characterized to be antiporters that mediate the transport of substrates in exchange for protons. In plants, alkaloids, phytohormones, ion chelators, and flavonoids have been reported to be the substrates of MATE transporters. Structural analyses have been conducted to dissect the functional significance of various motifs of MATE proteins. However, an understanding of the functions of the N- and C-termini has been inadequate. Here, by performing phylogenetic analyses and protein sequence alignment of 14 representative plant species, we identified a distinctive N-terminal poly-glutamate motif among a cluster of MATE proteins in soybean. Amongst them, GmMATE4 has the most consecutive glutamate residues at the N-terminus. A subcellular localization study showed that GmMATE4 was localized at the vacuolar membrane-like structure. Protein charge prediction showed that the mutation of the glutamate residues to alanine would reduce the negative charge at the N-terminus. Using yeast as the model, we showed that GmMATE4 mediated the transport of daidzein, genistein, glycitein, and glycitin. In addition, the glutamate-to-alanine mutation reduced the isoflavone transport capacity of GmMATE4. Altogether, we demonstrated GmMATE4 as an isoflavone transporter and the functional significance of the N-terminal poly-glutamate motif of GmMATE4 for regulating the isoflavone transport activity.

The Identification of MATE Antisense Transcripts in Soybean Using Strand-Specific RNA-Seq Datasets.

Natural antisense transcripts (NATs) have been generally reported as negative regulators of their sense counterparts. Multidrug and toxic compound extrusion (MATE) proteins mediate the transport of various substrates. Although MATEs have been identified genome-wide in various plant species, their transcript regulators remain unclear. Here, using the publicly available strand-specific RNA-seq datasets of Glycine soja (wild soybean) which have the data from various tissues including developing pods, developing seeds, embryos, cotyledons and hypocotyls, roots, apical buds, stems, and flowers, we identified 35 antisense transcripts of MATEs from 28 gene loci after transcriptome assembly. Spearman correlation coefficients suggested the positive expression correlations of eight MATE antisense and sense transcript pairs. By aligning the identified transcripts with the reference genome of Glycine max (cultivated soybean), the MATE antisense and sense transcript pairs were identified. Using soybean C08 (Glycine max), in developing pods and seeds, the positive correlations between MATE antisense and sense transcript pairs were shown by RT-qPCR. These findings suggest that soybean antisense transcripts are not necessarily negative transcription regulators of their sense counterparts. This study enhances the existing knowledge on the transcription regulation of MATE transporters by uncovering the previously unknown MATE antisense transcripts and their potential synergetic effects on sense transcripts.

The structure of the Aquifex aeolicus MATE family multidrug resistance transporter and sequence comparisons suggest the existence of a new subfamily

Multidrug and toxic compound extrusion (MATE) transporters are widespread in all domains of life. Bacterial MATE transporters confer multidrug resistance by utilizing an electrochemical gradient of H+ or Na+ to export xenobiotics across the membrane. Despite the availability of X-ray structures of several MATE transporters, a detailed understanding of the transport mechanism has remained elusive. Here we report the crystal structure of a MATE transporter from Aquifex aeolicus at 2.0-Å resolution. In light of its phylogenetic placement outside of the diversity of hitherto-described MATE transporters and the lack of conserved acidic residues, this protein may represent a subfamily of prokaryotic MATE transporters, which was proven by phylogenetic analysis. Furthermore, the crystal structure and substrate docking results indicate that the substrate binding site is located in the N bundle. The importance of residues surrounding this binding site was demonstrated by structure-based site-directed mutagenesis. We suggest that Aq_128 is functionally similar but structurally diverse from DinF subfamily transporters. Our results provide structural insights into the MATE transporter, which further advances our global understanding of this important transporter family.

Salt Stress-Induced Anthocyanin Biosynthesis Genes and MATE Transporter Involved in Anthocyanin Accumulation in Daucus carota Cell Culture.

Anthocyanins biosynthesis is a well-studied biosynthesis pathway in Daucus carota. However, the scale-up production at the bioreactor level and transporter involved in accumulation is poorly understood. To increase anthocyanin content and elucidate the molecular mechanism involved in accumulation, we examined D. carota cell culture in flask and bioreactor for 18 days under salt stress (20.0 mM NH4NO3/37.6 mM KNO3) at 3 day intervals. The expression of anthocyanin biosynthesis and putative MATE (multidrug and toxic compound extrusion) transporter expression was analyzed by qRT-PCR. It was observed that there was a significant enhancement of anthocyanin in the bioreactor compared to the control culture. A correlation was observed between the expression of MATE and the anthocyanin biosynthesis genes (CHS, C4H, LDOX, and UFGT) on the 9th day in a bioreactor, where maximum anthocyanin accumulation and expression was detected. We hypothesize the involvement of MATE in transporting anthocyanin to tonoplast in D. carota culture under salt stress.

Genome-wide identification, characterization and expression analysis of MATE family genes in apple (Malus \xd7 domestica Borkh)

BackgroundAs an important group of the multidrug efflux transporter family, the multidrug and toxic compound extrusion (MATE) family has a wide range of functions and is distributed in all kingdoms of living organisms. However, only two MATE genes in apple have been analyzed and genome-wide comprehensive analysis of MATE family is needed.ResultsIn this study, a total of 66 MATE (MdMATE) candidates encoding putative MATE transporters were identified in the apple genome. These MdMATE genes were classified into four groups by phylogenetic analysis with MATE genes in Arabidopsis. Synteny analysis reveals that whole genome duplication (WGD) and segmental duplication events played a major role in the expansion of MATE gene family in apple. MdMATE genes show diverse expression patterns in different tissues/organs and developmental stages. Analysis of cis-regulatory elements in MdMATE promoter regions indicates that the function of MdMATE genes is mainly related to stress response. Besides, the changes of gene expression levels upon different pathogen infections reveal that MdMATE genes are involved in biotic stress response.ConclusionsIn this work, we systematically identified MdMATE genes in apple genome using a set of bioinformatics approaches. Our comprehensive analysis provided valuable resources for improving disease resistance in apple and further functional characterization of MATE genes in other species.

Crystal structures of a nicotine MATE transporter provide insight into its mechanism of substrate transport.

A transporter of the multidrug and toxic compound extrusion (MATE) family, Nicotianatabacum MATE2 (NtMATE2), is located in the vacuole membrane of the tobacco plant root and is involved in the transportation of nicotine, a secondary or specialized metabolic compound in Solanaceae. Here, we report the crystal structures of NtMATE2 in its outward-facing forms. The overall structure has a bilobate V-shape with pseudo-symmetrical assembly of the N- and C-lobes. In one crystal structure, the C-lobe cavity of NtMATE2 interacts with an unidentified molecule that may partially mimic a substrate. In addition, NtMATE2-specific conformational transitions imply that an unprecedented movement of the transmembrane α-helix 7 is related to the release of the substrate into the vacuolar lumen.

Engineered MATE multidrug transporters reveal two functionally distinct ion-coupling pathways in NorM from Vibrio cholerae

Multidrug and toxic compound extrusion (MATE) transport proteins confer multidrug resistance on pathogenic microorganisms and affect pharmacokinetics in mammals. Our understanding of how MATE transporters work, has mostly relied on protein structures and MD simulations. However, the energetics of drug transport has not been studied in detail. Many MATE transporters utilise the electrochemical H+ or Na+ gradient to drive substrate efflux, but NorM-VC from Vibrio cholerae can utilise both forms of metabolic energy. To dissect the localisation and organisation of H+ and Na+ translocation pathways in NorM-VC we engineered chimaeric proteins in which the N-lobe of H+-coupled NorM-PS from Pseudomonas stutzeri is fused to the C-lobe of NorM-VC, and vice versa. Our findings in drug binding and transport experiments with chimaeric, mutant and wildtype transporters highlight the versatile nature of energy coupling in NorM-VC, which enables adaptation to fluctuating salinity levels in the natural habitat of V. cholerae.

Conserved binding site in the N-lobe of prokaryotic MATE transporters suggests a role for Na+ in ion-coupled drug efflux.

In both prokaryotes and eukaryotes, multidrug and toxic-compound extrusion (MATE) transporters catalyze the efflux of a broad range of cytotoxic compounds, including human-made antibiotics and anticancer drugs. MATEs are secondary-active antiporters, i.e. their drug-efflux activity is coupled to, and powered by, the uptake of ions down a pre-existing transmembrane electrochemical gradient. Key aspects of this mechanism, however, remain to be delineated, such as its ion specificity and stoichiometry. We previously revealed the existence of a Na+-binding site in a MATE transporter from Pyroccocus furiosus (PfMATE) and hypothesized that this site might be broadly conserved among prokaryotic MATEs. Here, we evaluate this hypothesis by analyzing VcmN and ClbM, which along with PfMATE are the only three prokaryotic MATEs whose molecular structures have been determined at resolutions better than 3 Å. Analysis of available crystallographic data and molecular dynamics simulations indeed reveal an occupied Na+-binding site in the N-terminal lobe of both structures, analogous to that identified in PfMATE. We likewise find this site to be strongly selective against K+, suggesting it is mechanistically significant. Consistent with these computational results, DEER spectroscopy measurements for multiple doubly-spin-labeled VcmN constructs demonstrate Na+-dependent changes in protein conformation. The existence of this binding site in three MATE orthologs implicates Na+ in the ion-coupled drug-efflux mechanisms of this class of transporters. These results also imply that observations of H+-dependent activity stem either from a site elsewhere in the structure, or from H+ displacing Na+ under certain laboratory conditions, as has been noted for other Na+-driven transport systems.

Conserved binding site in the N-lobe of prokaryotic MATE transporters suggests a role for Na+ in ion-coupled drug efflux

In both prokaryotes and eukaryotes, multidrug and toxic-compound extrusion (MATE) transporters catalyze the efflux of a broad range of cytotoxic compounds, including human-made antibiotics and anticancer drugs. MATEs are secondary-active antiporters, i.e., their drug-efflux activity is coupled to, and powered by, the uptake of ions down a preexisting transmembrane electrochemical gradient. Key aspects of this mechanism, however, remain to be delineated, such as its ion specificity and stoichiometry. We previously revealed the existence of a Na+-binding site in a MATE transporter from Pyroccocus furiosus (PfMATE) and hypothesized that this site might be broadly conserved among prokaryotic MATEs. Here, we evaluate this hypothesis by analyzing VcmN and ClbM, which along with PfMATE are the only three prokaryotic MATEs whose molecular structures have been determined at atomic resolution, i.e. better than 3 Å. Reinterpretation of existing crystallographic data and molecular dynamics simulations indeed reveal an occupied Na+-binding site in the N-terminal lobe of both structures, analogous to that identified in PfMATE. We likewise find this site to be strongly selective against K+, suggesting it is mechanistically significant. Consistent with these computational results, DEER spectroscopy measurements for multiple doubly-spin-labeled VcmN constructs demonstrate Na+-dependent changes in protein conformation. The existence of this binding site in three MATE orthologs implicates Na+ in the ion-coupled drug-efflux mechanisms of this class of transporters. These results also imply that observations of H+-dependent activity likely stem either from a site elsewhere in the structure, or from H+ displacing Na+ under certain laboratory conditions, as has been noted for other Na+-driven transport systems.