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Zinc transporters belonging to the Cation Diffusion Facilitator (CDF) family have complementary roles in transporting zinc out of the cytosol.

ABSTRACT: Zinc is an essential trace element that is required for the function of a large number of proteins. As these zinc-binding proteins are found within the cytosol and organelles, all eukaryotes require mechanisms to ensure that zinc is delivered to organelles, even under conditions of zinc deficiency. Although many zinc transporters belonging to the Cation Diffusion Facilitator (CDF) families have well characterized roles in transporting zinc into the lumens of intracellular compartments, relatively little is known about the mechanisms that maintain organelle zinc homeostasis. The fission yeast Schizosaccharomyces pombe is a useful model system to study organelle zinc homeostasis as it expresses three CDF family members that transport zinc out of the cytosol into intracellular compartments: Zhf1, Cis4, and Zrg17. Zhf1 transports zinc into the endoplasmic reticulum, and Cis4 and Zrg17 form a heterodimeric complex that transports zinc into the cis-Golgi. Here we have used the high and low affinity ZapCY zinc-responsive FRET sensors to examine cytosolic zinc levels in yeast mutants that lack each of these CDF proteins. We find that deletion of cis4 or zrg17 leads to higher levels of zinc accumulating in the cytosol under conditions of zinc deficiency, whereas deletion of zhf1 results in zinc accumulating in the cytosol when zinc is not limiting. We also show that the expression of cis4, zrg17, and zhf1 is independent of cellular zinc status. Taken together our results suggest that the Cis4/Zrg17 complex is necessary for zinc transport out of the cytosol under conditions of zinc-deficiency, while Zhf1 plays the dominant role in removing zinc from the cytosol when labile zinc is present. We propose that the properties and/or activities of individual CDF family members are fine-tuned to enable cells to control the flux of zinc out of the cytosol over a broad range of environmental zinc stress.

SUBMITTER: Choi S

PROVIDER: S-EPMC5864093 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe 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.ResultsRepresentative 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.ConclusionIn 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:BackgroundMembers of the cation diffusion facilitator (CDF) family are integral membrane divalent cation transporters that transport metal ions out of the cytoplasm either into the extracellular space or into internal compartments such as the vacuole. The spectrum of cations known to be transported by proteins of the CDF family include Zn, Fe, Co, Cd, and Mn. Members of this family have been identified in prokaryotes, eukaryotes, and archaea, and in sequenced plant genomes. CDF families range in size from nine members in Selaginella moellendorffii to 19 members in Populus trichocarpa. Phylogenetic analysis suggests that the CDF family has expanded within plants, but a definitive plant CDF family phylogeny has not been constructed.ResultsRepresentative CDF members were annotated from diverse genomes across the Viridiplantae and Rhodophyta lineages and used to identify phylogenetic relationships within the CDF family. Bayesian phylogenetic analysis of CDF amino acid sequence data supports organizing land plant CDF family sequences into 7 groups. The origin of the 7 groups predates the emergence of land plants. Among these, 5 of the 7 groups are likely to have originated at the base of the tree of life, and 2 of 7 groups appear to be derived from a duplication event prior to or coincident with land plant evolution. Within land plants, local expansion continues within select groups, while several groups are strictly maintained as one gene copy per genome.ConclusionsDefining the CDF gene family phylogeny contributes to our understanding of this family in several ways. First, when embarking upon functional studies of the members, defining primary groups improves the predictive power of functional assignment of orthologous/paralogous genes and aids in hypothesis generation. Second, defining groups will allow a group-specific sequence motif to be generated that will help define future CDF family sequences and aid in functional motif identification, which currently is lacking for this family in plants. Third, the plant-specific expansion resulting in Groups 8 and 9 evolved coincident to the early primary radiation of plants onto land, suggesting these families may have been important for early land colonization.

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:BACKGROUND:Hexaploid wheat is an important cereal crop that has been targeted to enhance grain micronutrient content including zinc (Zn) and iron (Fe). In this direction, modulating the expression of plant transporters involved in Fe and Zn homeostasis has proven to be one of the promising approaches. The present work was undertaken to identify wheat zinc-induced facilitator-like (ZIFL) family of transporters. The wheat ZIFL genes were characterized for their transcriptional expression response during micronutrient fluctuations and exposure to multiple heavy metals. RESULTS:The genome-wide analyses resulted in identification of fifteen putative TaZIFL-like genes, which were distributed only on Chromosome 3, 4 and 5. Wheat ZIFL proteins subjected to the phylogenetic analysis showed the uniform distribution along with rice, Arabidopsis and maize. In-silico analysis of the promoters of the wheat ZIFL genes demonstrated the presence of multiple metal binding sites including those which are involved in Fe and heavy metal homeostasis. Quantitative real-time PCR analysis of wheat ZIFL genes suggested the differential regulation of the transcripts in both roots and shoots under Zn surplus and also during Fe deficiency. Specifically, in roots, TaZIFL2.3, TaZIFL4.1, TaZIFL4.2, TaZIFL5, TaZIFL6.1 and TaZIFL6.2 were significantly up-regulated by both Zn and Fe. This suggested that ZIFL could possibly be regulated by both the nutrient stress in a tissue specific manner. When exposed to heavy metals, TaZIFL4.2 and TaZIFL7.1 show significant up-regulation, whereas TaZIFL5 and TaZIFL6.2 remained almost unaffected. CONCLUSION:This is the first report for detailed analysis of wheat ZIFL genes. ZIFL genes also encode for transporter of mugineic acid (TOM) proteins, that are involved in the release of phytosiderophores to enhance Fe/Zn uptake. The detailed expression analysis suggests the varying expression patterns during development of wheat seedlings and also against abiotic/biotic stresses. Overall, this study will lay foundation to prioritize functional assessment of the candidate ZIFL as a putative TOM protein in wheat.

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Zinc transporters belonging to the Cation Diffusion Facilitator (CDF) family have complementary roles in transporting zinc out of the cytosol.

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