OsMTP11 is localised at the Golgi and contributes to Mn tolerance.
ABSTRACT: Membrane transporters play a key role in obtaining sufficient quantities of manganese (Mn) but also in protecting against Mn toxicity. We have characterized OsMTP11, a member of the Cation Diffusion Facilitator/Metal Tolerance Protein (CDF/MTP) family of metal cation transporters in Oryza sativa. We demonstrate that OsMTP11 functions in alleviating Mn toxicity as its expression can rescue the Mn-sensitive phenotype of the Arabidopsis mtp11-3 knockout mutant. When expressed stably in Arabidopsis and transiently in rice and tobacco, it localises to the Golgi. OsMTP11 partially rescues the Mn-hypersensitivity of the pmr1 yeast mutant but only slightly alleviates the Zn sensitivity of the zrc1 cot1 yeast mutant. Overall, these results suggest that OsMTP11 predominantly functions as a Mn-transporting CDF with lower affinity for Zn. Site-directed mutagenesis studies revealed four substitutions in OsMTP11 that appear to alter its transport activity. OsMTP11 harbouring a substitution of leucine 150 to a serine fully rescued pmr1 Mn-sensitivity at all concentrations tested. The other substitutions, including those at conserved DxxxD domains, reduced complementation of pmr1 to different levels. This indicates their importance for OsMTP11 function and is a starting point for refining transporter activity/specificity.
Project description:Heavy metal homeostasis is maintained in plant cells by specialized transporters which compartmentalize or efflux metal ions, maintaining cytosolic concentrations within a narrow range. OsMTP1 is a member of the cation diffusion facilitator (CDF)/metal tolerance protein (MTP) family of metal cation transporters in Oryza sativa, which is closely related to Arabidopsis thaliana MTP1. Functional complementation of the Arabidopsis T-DNA insertion mutant mtp1-1 demonstrates that OsMTP1 transports Zn in planta and localizes at the tonoplast. When heterologously expressed in the yeast mutant zrc1 cot1, OsMTP1 complemented its Zn hypersensitivity and was also localized to the vacuole. OsMTP1 alleviated, to some extent, the Co sensitivity of this mutant, rescued the Fe hypersensitivity of the ccc1 mutant at low Fe concentrations, and restored growth of the Cd-hypersensitive mutant ycf1 at low Cd concentrations. These results suggest that OsMTP1 transports Zn but also Co, Fe, and Cd, possibly with lower affinity. Site-directed mutagenesis studies revealed two substitutions in OsMTP1 that alter the transport function of this protein. OsMTP1 harbouring a substitution of Leu82 to a phenylalanine can still transport low levels of Zn, with an enhanced affinity for Fe and Co, and a gain of function for Mn. A substitution of His90 with an aspartic acid completely abolishes Zn transport but improves Fe transport in OsMTP1. These amino acid residues are important in determining substrate specificity and may be a starting point for refining transporter activity in possible biotechnological applications, such as biofortification and phytoremediation.
Project description:The ability of Thlaspi goesingense to hyperaccumulate Ni seems to be governed in part by enhanced accumulation of Ni within leaf vacuoles. We have characterized genes from T. goesingense encoding putative vacuolar metal ion transport proteins, termed metal tolerance proteins (TgMTPs). These proteins contain all of the features of cation-efflux family members, and evidence indicates they are derived from a single genomic sequence (TgMTP1) that gives rise to an unspliced (TgMTP1t1) and a spliced (TgMTP1t2) transcript. Heterologous expression of these transcripts in yeast lacking the TgMTP1 orthologues COT1 and ZRC1 complements the metal sensitivity of these yeast strains, suggesting that TgMTP1s are able to transport metal ions into the yeast vacuole in a manner similar to COT1 and ZRC1. The unspliced and spliced TgMTP1 variants differ within a histidine-rich putative metal-binding domain, and these sequence differences are reflected as alterations in the metal specificities of these metal ion transporters. When expressed in yeast, TgMTP1t1 confers the highest level of tolerance to Cd, Co, and Zn, whereas TgMTP1t2 confers the highest tolerance to Ni. TgMTP1 transcripts are highly expressed in T. goesingense compared with orthologues in the nonaccumulators Arabidopsis thaliana, Thlaspi arvense, and Brassica juncea. We propose that the high-level expression of TgMTP1 in T. goesingense accounts for the enhanced ability of this hyperaccumulator to accumulate metal ions within shoot vacuoles.
Project description:In the cation diffusion facilitator (CDF) family, the transported substrates are confined to divalent metal ions, such as Zn2+, Fe2+, and Mn2+. However, this study identifies a novel CDF member designated MceT from the moderate halophile Planococcus dechangensis. MceT functions as a Na+(Li+, K+)/H+ antiporter, together with its capability of facilitated Zn2+ diffusion into cells, which have not been reported in any identified CDF transporters as yet. MceT is proposed to represent a novel CDF group, Na-CDF, which shares significantly distant phylogenetic relationship with three known CDF groups including Mn-CDF, Fe/Zn-CDF, and Zn-CDF. Variation of key function-related residues to "Y44-S48-Q150" in two structural motifs explains a significant discrimination in cation selectivity between Na-CDF group and three major known CDF groups. Functional analysis via site-directed mutagenesis confirms that MceT employs Q150, S158, and D184 for the function of MceT as a Na+(Li+, K+)/H+ antiporter, and retains D41, D154, and D184 for its facilitated Zn2+ diffusion into cells. These presented findings imply that MceT has evolved from its native CDF family function to a Na+/H+ antiporter in an evolutionary strategy of the substitution of key conserved residues to "Q150-S158-D184" motif. More importantly, the discovery of MceT contributes to a typical transporter model of CDF family with the unique structural motifs, which will be utilized to explore the cation-selective mechanisms of secondary transporters.
Project description:Cation diffusion facilitators (CDFs) are a large family of divalent metal transporters that collectively possess broad metal specificity and contribute to intracellular metal homeostasis and virulence in bacterial pathogens. Streptococcus pneumoniae expresses two homologous CDF efflux transporters, MntE and CzcD. Cells lacking mntE or czcD are sensitive to manganese (Mn) or zinc (Zn) toxicity, respectively, and specifically accumulate Mn or Zn, respectively, thus suggesting that MntE selectively transports Mn, while CzcD transports Zn. Here, we probe the origin of this metal specificity using a phenotypic growth analysis of pneumococcal variants. Structural homology to Escherichia coli YiiP predicts that both MntE and CzcD are dimeric and each protomer harbors four pairs of conserved metal-binding sites, termed the A site, the B site, and the C1/C2 binuclear site. We find that single amino acid mutations within both the transmembrane domain A site and the B site in both CDFs result in a cellular metal sensitivity similar to that of the corresponding null mutants. However, multiple mutations in the predicted cytoplasmic C1/C2 cluster of MntE have no impact on cellular Mn resistance, in contrast to the analogous substitutions in CzcD, which do have on impact on cellular Zn resistance. Deletion of the MntE-specific C-terminal tail, present only in Mn-specific bacterial CDFs, resulted in only a modest growth phenotype. Further analysis of MntE-CzcD functional chimeric transporters showed that Asn and Asp in the ND-DD A-site motif of MntE and the most N-terminal His in the HD-HD site A of CzcD (the specified amino acids are underlined) play key roles in transporter metal selectivity.Cation diffusion facilitator (CDF) proteins are divalent metal ion transporters that are conserved in organisms ranging from bacteria to humans and that play important roles in cellular physiology, from metal homeostasis and resistance to type I diabetes in vertebrates. The respiratory pathogen Streptococcus pneumoniae expresses two metal CDF transporters, CzcD and MntE. How CDFs achieve metal selectivity is unclear. We show here that CzcD and MntE are true paralogs, as CzcD transports zinc, while MntE selectively transports manganese. Through the use of an extensive collection of pneumococcal variants, we show that a primary determinant for metal selectivity is the A site within the transmembrane domain. This extends our understanding of how CDFs discriminate among transition metals.
Project description:Arabidopsis thaliana AtMTP1 belongs to the cation diffusion facilitator family and is localized on the vacuolar membrane. We investigated the enzymatic kinetics of AtMTP1 by a heterologous expression system in the yeast Saccharomyces cerevisiae, which lacked genes for vacuolar membrane zinc transporters ZRC1 and COT1. The yeast mutant expressing AtMTP1 heterologously was tolerant to 10 mm ZnCl(2). Active transport of zinc into vacuoles of living yeast cells expressing AtMTP1 was confirmed by the fluorescent zinc indicator FuraZin-1. Zinc transport was quantitatively analyzed by using vacuolar membrane vesicles prepared from AtMTP1-expressing yeast cells and radioisotope (65)Zn(2+). Active zinc uptake depended on a pH gradient generated by endogenous vacuolar H(+)-ATPase. The activity was inhibited by bafilomycin A(1), an inhibitor of the H(+)-ATPase. The K(m) for Zn(2+) and V(max) of AtMTP1 were determined to be 0.30 microm and 1.22 nmol/min/mg, respectively. We prepared a mutant AtMTP1 that lacked the major part (32 residues from 185 to 216) of a long histidine-rich hydrophilic loop in the central part of AtMTP1. Yeast cells expressing the mutant became hyperresistant to high concentrations of Zn(2+) and resistant to Co(2+). The K(m) and V(max) values were increased 2-11-fold. These results indicate that AtMTP1 functions as a Zn(2+)/H(+) antiporter in vacuoles and that a histidine-rich region is not essential for zinc transport. We propose that a histidine-rich loop functions as a buffering pocket of Zn(2+) and a sensor of the zinc level at the cytoplasmic surface. This loop may be involved in the maintenance of the level of cytoplasmic Zn(2+).
Project description:BACKGROUND: The Cation Diffusion Facilitator (CDF) family is a ubiquitous family of heavy metal transporters. Much interest in this family has focused on implications for human health and bioremediation. In this work a broad phylogenetic study has been undertaken which, considered in the context of the functional characteristics of some fully characterised CDF transporters, has aimed at identifying molecular determinants of substrate selectivity and at suggesting metal specificity for newly identified CDF transporters. RESULTS: Representative CDF members from all three kingdoms of life (Archaea, Eubacteria, Eukaryotes) were retrieved from genomic databases. Protein sequence alignment has allowed detection of a modified signature that can be used to identify new hypothetical CDF members. Phylogenetic reconstruction has classified the majority of CDF family members into three groups, each containing characterised members that share the same specificity towards the principally-transported metal, i.e. Zn, Fe/Zn or Mn. The metal selectivity of newly identified CDF transporters can be inferred by their position in one of these groups. The function of some conserved amino acids was assessed by site-directed mutagenesis in the poplar Zn2+ transporter PtdMTP1 and compared with similar experiments performed in prokaryotic members. An essential structural role can be assigned to a widely conserved glycine residue, while aspartate and histidine residues, highly conserved in putative transmembrane domains, might be involved in metal transport. The potential role of group-conserved amino acid residues in metal specificity is discussed. CONCLUSION: In the present study phylogenetic and functional analyses have allowed the identification of three major substrate-specific CDF groups. The metal selectivity of newly identified CDF transporters can be inferred by their position in one of these groups. The modified signature sequence proposed in this work can be used to identify new hypothetical CDF members.
Project description:Members of the cation diffusion facilitator (CDF) family have been identified in all kingdoms of life. They have been divided into three subgroups, namely Zn-CDF, Fe/Zn-CDF, and Mn-CDF, based on their putative specificity to transported metal ions. The plant metal tolerance protein 6 (MTP6) proteins fall into the Fe/Zn-CDF subgroup; however, their function in iron/zinc transport has not yet been confirmed. Here, we characterized the MTP6 protein from cucumber, Cucumis sativus. When expressed in yeast and in protoplasts isolated from Arabidopsis cells, CsMTP6 localized in mitochondria and contributed to the efflux of Fe and Mn from these organelles. Immunolocalization of CsMTP6 in cucumber membranes confirmed this association with mitochondria. Root expression and protein levels of CsMTP6 were significantly up-regulated in conditions of Fe deficiency and excess, but were not affected by Mn availability. These results indicate that MTP6 proteins contribute to the distribution of Fe and Mn between the cytosol and mitochondria of plant cells, and are regulated by Fe to maintain mitochondrial and cytosolic iron homeostasis under varying conditions of Fe availability.
Project description:Zinc homeostasis was investigated in Nostoc punctiforme. Cell tolerance to Zn(2+) over 14 days showed that ZnCl(2) levels above 22 microM significantly reduced cell viability. After 3 days in 22 microM ZnCl(2), ca. 12% of the Zn(2+) was in an EDTA-resistant component, suggesting an intracellular localization. Zinquin fluorescence was detected within cells exposed to concentrations up to 37 microM relative to 0 microM treatment. Radiolabeled (65)Zn showed Zn(2+) uptake increased over a 3-day period, while efflux occurred more rapidly within a 3-h time period. Four putative genes involved in Zn(2+) uptake and efflux in N. punctiforme were identified: (i) the predicted Co/Zn/Cd cation transporter, putative CDF; (ii) the predicted divalent heavy-metal cation transporter, putative Zip; (iii) the ATPase component and Fe/Zn uptake regulation protein, putative Fur; and (iv) an ABC-type Mn/Zn transport system, putative zinc ZnuC, ZnuABC system component. Quantitative real-time PCR indicated the responsiveness of all four genes to 22 microM ZnCl(2) within 3 h, followed by a reduction to below basal levels after 24 h by putative ZIP, ZnuC, and Fur and a reduction to below basal level after 72 h by putative CDF efflux gene. These results demonstrate differential regulation of zinc transporters over time, indicating a role for them in zinc homeostasis in N. punctiforme.
Project description:Cation diffusion facilitator transporters are found in all three Kingdoms of life and are involved in transporting transition metals out of the cytosol. The metals they transport include Zn2+, Co2+, Fe2+, Cd2+, Ni2+ and Mn2+; however, no single transporter transports all metals. Previously we showed that a single amino acid mutation in the yeast vacuolar zinc transporter Zrc1 changed its substrate specificity from Zn2+ to Fe2+ and Mn2+ [Lin, Kumanovics, Nelson, Warner, Ward and Kaplan (2008) J. Biol. Chem. 283, 33865-33873]. Mutant Zrc1 that gained iron transport activity could protect cells with a deletion in the vacuolar iron transporter (CCC1) from high iron toxicity. Utilizing suppression of high iron toxicity and PCR mutagenesis of ZRC1, we identified other amino acid substitutions within ZRC1 that changed its metal specificity. All Zrc1 mutants that transported Fe2+ could also transport Mn2+. Some Zrc1 mutants lost the ability to transport Zn2+, but others retained the ability to transport Zn2+. All of the amino acid substitutions that resulted in a gain in Fe2+ transport activity were found in transmembrane domains. In addition to alteration of residues adjacent to the putative metal- binding site in two transmembrane domains, alteration of residues distant from the binding site affected substrate specificity. These results suggest that substrate selection involves co-operativity between transmembrane domains.
Project description:Metal tolerance proteins (MTPs) are plant members of the cation diffusion facilitator (CDF) transporter family involved in cellular metal homeostasis. Members of the CDF family are ubiquitously found in all living entities and show principal selectivity for Zn(2+), Mn(2+), and Fe(2+). Little is known regarding metal selectivity determinants of CDFs. We identified a novel cereal member of CDFs in barley, termed HvMTP1, that localizes to the vacuolar membrane. Unlike its close relative AtMTP1, which is highly selective for Zn(2+), HvMTP1 exhibits selectivity for both Zn(2+) and Co(2+) as assessed by its ability to suppress yeast mutant phenotypes for both metals. Expression of HvMTP1/AtMTP1 chimeras in yeast revealed a five-residue sequence within the AtMTP1 N-segment of the His-rich intracytoplasmic loop that confines specificity to Zn(2+). Furthermore, mutants of AtMTP1 generated through random mutagenesis revealed residues embedded within transmembrane domain 3 that additionally specify the high degree of Zn(2+) selectivity. We propose that the His-rich loop, which might play a role as a zinc chaperone, determines the identity of the metal ions that are transported. The residues within transmembrane domain 3 can also influence metal selectivity, possibly through conformational changes induced at the cation transport site located within the membrane or at the cytoplasmic C-terminal domain.