In vitro reconstitution of transition metal transporters.

Transition metal ions are critically important across all kingdoms of life. The chemical properties of iron, copper, zinc, manganese, cobalt, and nickel make them very attractive for use as cofactors in metalloenzymes and/or metalloproteins. Their versatile chemistry in aqueous solution enables them to function both as electron donors and acceptors, and thus participate in both reduction and oxidation reactions respectively. Transition metal ions can also function as nonredox multidentate coordination sites that play essential roles in macromolecular structure and function. Malfunction in transition metal transport and homeostasis has been linked to a wide number of human diseases including cancer, diabetes, and neurodegenerative disorders. Transition metal transporters are central players in the physiology of transition metals whereby they move transition metals in and out of cellular compartments. In this review, we provide a comprehensive overview of in vitro reconstitution of the activity of integral membrane transition metal transporters and discuss strategies that have been successfully implemented to overcome the challenges. We also discuss recent advances in our understanding of transition metal transport mechanisms and the techniques that are currently used to decipher the molecular basis of transport activities of these proteins. Deep mechanistic insights into transition metal transport systems will be essential to understand their malfunction in human diseases and target them for potential therapeutic strategies.

Reduced expression of bZIP19 and bZIP23 increases zinc and cadmium accumulation in Arabidopsis halleri.

Zinc is an essential micronutrient for all living organisms. When challenged by zinc-limiting conditions, Arabidopsis thaliana plants use a strategy centered on two transcription factors, bZIP19 and bZIP23, to enhance the expression of several zinc transporters to improve their zinc uptake capacity. In the zinc and cadmium hyperaccumulator plant Arabidopsis halleri, highly efficient root-to-shoot zinc translocation results in constitutive local zinc deficiency in roots and in constitutive high expression of zinc deficiency-responsive ZIP genes, supposedly boosting zinc uptake and accumulation. Here, to disrupt this process and to analyze the functions of AhbZIP19, AhbZIP23 and their target genes in hyperaccumulation, the genes encoding both transcriptional factors were knocked down using artificial microRNAs (amiRNA). Although AhbZIP19, AhbZIP23, and their ZIP target genes were downregulated, amiRNA lines surprisingly accumulated more zinc and cadmium compared to control lines in both roots and shoot driving to shoot toxicity symptoms. These observations suggested the existence of a substitute metal uptake machinery in A. halleri to maintain hyperaccumulation. We propose that the iron uptake transporter AhIRT1 participates in this alternative pathway in A. halleri.

The Role of Low-Molecular-Weight Organic Acids in Metal Homeostasis in Plants.

Low-molecular-weight organic acids (LMWOAs) are essential O-containing metal-binding ligands involved in maintaining metal homeostasis, various metabolic processes, and plant responses to biotic and abiotic stress. Malate, citrate, and oxalate play a crucial role in metal detoxification and transport throughout the plant. This review provides a comparative analysis of the accumulation of LMWOAs in excluders, which store metals mainly in roots, and hyperaccumulators, which accumulate metals mainly in shoots. Modern concepts of the mechanisms of LMWOA secretion by the roots of excluders and hyperaccumulators are summarized, and the formation of various metal complexes with LMWOAs in the vacuole and conducting tissues, playing an important role in the mechanisms of metal detoxification and transport, is discussed. Molecular mechanisms of transport of LMWOAs and their complexes with metals across cell membranes are reviewed. It is discussed whether different endogenous levels of LMWOAs in plants determine their metal tolerance. While playing an important role in maintaining metal homeostasis, LMWOAs apparently make a minor contribution to the mechanisms of metal hyperaccumulation, which is associated mainly with root exudates increasing metal bioavailability and enhanced xylem loading of LMWOAs. The studies of metal-binding compounds may also contribute to the development of approaches used in biofortification, phytoremediation, and phytomining.

Cation diffusion facilitator proteins of Beta vulgaris reveal diversity of metal handling in dicotyledons.

Manganese (Mn), iron (Fe), and zinc (Zn) are essential for diverse processes in plants, but their availability is often limiting or excessive. Cation diffusion facilitator (CDF) proteins have been implicated in the allocation of those metals in plants, whereby most of our mechanistic understanding has been obtained in Arabidopsis. It is unclear to what extent this can be generalized to other dicots. We characterized all CDFs/metal tolerance proteins of sugar beet (Beta vulgaris spp. vulgaris), which is phylogenetically distant from Arabidopsis. Analysis of subcellular localization, substrate selectivities, and transcriptional regulation upon exposure to metal deficiencies and toxicities revealed unexpected deviations from their Arabidopsis counterparts. Localization and selectivity of some members were modulated by alternative splicing. Notably, unlike in Arabidopsis, Mn- and Zn-sequestrating members were not induced in Fe-deficient roots, pointing to differences in the Fe acquisition machinery. This was supported by low Zn and Mn accumulation under Fe deficiency and a strikingly increased Fe accumulation under Mn and Zn excess, coinciding with an induction of BvIRT1. High Zn load caused a massive upregulation of Zn-BvMTPs. The results suggest that the employment of the CDF toolbox is highly diverse amongst dicots, which questions the general applicability of metal homeostasis models derived from Arabidopsis.

Compositional variations in metal nanoparticle components of welding fumes impact lung epithelial cell toxicity.

Welding fumes contain harmful metals and gas by-products associated with development of lung dysfunction, asthma, bronchitis, and lung cancer. Two prominent welding fume particulate metal components are nanosized iron (Fe) and manganese (Mn) which might induce oxidative stress and inflammation resulting in pulmonary injury. Welding fume toxicity may be dependent upon metal nanoparticle (NP) components. To examine toxicity of welding fume NP components, a system was constructed for controlled and continuous NP generation from commercial welding and customized electrodes with varying proportions of Fe and Mn. Aerosols generated consisted of nanosized particles and were compositionally consistent with each electrode. Human alveolar lung A459 epithelial cells were exposed to freshly generated metal NP mixtures at a target concentration of 100 µg/m3 for 6 hr and then harvested for assessment of cytotoxicity, generation of reactive oxygen species (ROS), and alterations in the expression of genes and proteins involved in metal regulation, inflammatory responses, and oxidative stress. Aerosol exposures decreased cell viability and induced increased ROS production. Assessment of gene expression demonstrated variable up-regulation in cellular mechanisms related to metal transport and storage, inflammation, and oxidative stress based upon aerosol composition. Specifically, interleukin-8 (IL-8) demonstrated the most robust changes in both transcriptional and protein levels after exposure. Interleukin-8 has been determined to serve as a primary cytokine mediating inflammatory responses induced by welding fume exposures in alveolar epithelial cells. Overall, this study demonstrated variations in cellular responses to metal NP mixtures suggesting compositional variations in NP content within welding fumes may influence inhalation toxicity.

Identification of a Czc-like operon of the periodontal pathobiont Porphyromonas gingivalis involved in metal ion efflux.

OBJECTIVE: The study aimed to investigate the role of the PGN2012 gene of the periodontitis contributing pathobiont Porphyromonas gingivalis. PGN2012 is a homolgue of TolC and is a gene our group previously showed was overexpressed in hyperinvasive cells. METHODS: The study used a combination of bioinformatics, knockout mutagenesis, growth experiments, biofilm assays and human cell invation assays to investigate PGN2012 function. RESULTS: Bioinformatics identified that PGN2012 is part of one of four TolC containing gene loci in P. gingivalis that we predicted may encode a metal resistance RND family tripartite pump, similar to those present in other Gram-negative bacteria, but which are not well understood in anaerobic bacteria. A ΔPGN2012 deletion displayed slightly reduced growth in liquid culture but did not effect biofilm formation or human cell invasion. When metal ions were included in the medium the mutant displayed significantly increased sensitivity to the divalent metal ions Zn2+ (500 µM), Co2+ (2 mM), and Cd2+(0.1 mM) but not Cu2+. CONCLUSIONS: We propose to rename the PGN2012-2014 genes czcCBA, which we suggest plays a role in intracellular stress resistance where zinc is often employed by host cells in antibacterial defence with implications for chronic infection in humans.

Nicotianamine: A Key Player in Metal Homeostasis and Hyperaccumulation in Plants.

Nicotianamine (NA) is a low-molecular-weight N-containing metal-binding ligand, whose accumulation in plant organs changes under metal deficiency or excess. Although NA biosynthesis can be induced in vivo by various metals, this non-proteinogenic amino acid is mainly involved in the detoxification and transport of iron, zinc, nickel, copper and manganese. This review summarizes the current knowledge on NA biosynthesis and its regulation, considers the mechanisms of NA secretion by plant roots, as well as the mechanisms of intracellular transport of NA and its complexes with metals, and its role in radial and long-distance metal transport. Its role in metal tolerance is also discussed. The NA contents in excluders, storing metals primarily in roots, and in hyperaccumulators, accumulating metals mainly in shoots, are compared. The available data suggest that NA plays an important role in maintaining metal homeostasis and hyperaccumulation mechanisms. The study of metal-binding compounds is of interdisciplinary significance, not only regarding their effects on metal toxicity in plants, but also in connection with the development of biofortification approaches to increase the metal contents, primarily of iron and zinc, in agricultural plants, since the deficiency of these elements in food crops seriously affects human health.

Arabidopsis thaliana Zn2+-efflux ATPases HMA2 and HMA4 are required for resistance to the necrotrophic fungus Plectosphaerella cucumerina BMM.

Zinc is an essential nutrient at low concentrations, but toxic at slightly higher ones. It has been proposed that hyperaccumulator plants may use the excess zinc to fend off pathogens and herbivores. However, there is little evidence of a similar response in other plants. Here we show that Arabidopsis thaliana leaves inoculated with the necrotrophic fungus Plectosphaerella cucumerina BMM (PcBMM) accumulate zinc and manganese at the infection site. Zinc accumulation did not occur in a double mutant in the zinc transporters HEAVY METAL ATPASE2 and HEAVY METAL ATPASE4 (HMA2 and HMA4), which has reduced zinc translocation from roots to shoots. Consistent with a role in plant immunity, expression of HMA2 and HMA4 was up-regulated upon PcBMM inoculation, and hma2hma4 mutants were more susceptible to PcBMM infection. This phenotype was rescued upon zinc supplementation. The increased susceptibility to PcBMM infection was not due to the diminished expression of genes involved in the salicylic acid, ethylene, or jasmonate pathways since they were constitutively up-regulated in hma2hma4 plants. Our data indicate a role of zinc in resistance to PcBMM in plants containing ordinary levels of zinc. This layer of immunity runs in parallel to the already characterized defence pathways, and its removal has a direct effect on resistance to pathogens.

A weak allele of OsNRAMP5 confers moderate cadmium uptake while avoiding manganese deficiency in rice.

Decreasing cadmium (Cd) concentrations in rice grains can effectively reduce potential risks to human health because rice is the major contributor to Cd intake in many diets. Among several genes involved in rice Cd accumulation, the loss of function of OsNRAMP5 is known to be effective in reducing grain concentration by inhibiting root uptake. However, disruption of this gene simultaneously decreases manganese (Mn) uptake because OsNRAMP5 is a major Mn transporter. With the aim of improving Mn uptake in OsNRAMP5 mutants while still restricting the grain Cd concentration below the upper limit of international standards, we identified a novel OsNRAMP5 allele encoding a protein in which glutamine (Q) at position 337 was replaced by lysine (K). The mutant carrying the OsNRAMP5-Q337K allele showed intermediate Cd and Mn accumulation between that of the wild-type and OsNRAMP5-knockout lines, and exhibited more resistance to Mn deficiency than the knockout lines. Different amino acid substitutions at position Q337 significantly affected the Cd and Mn transport activity in yeast cells, indicating that it is one of the crucial sites for OsNRAMP5 function. Our results suggest that the OsNRAMP5-Q337K allele might be useful for reducing grain Cd concentrations without causing severe Mn deficiency in rice cultivars through DNA marker-assisted breeding.

Identification and Characterization of ATP-Binding Cassette Transporters in Chlamydomonas reinhardtii.

Microalgae are promising microorganisms used to produce value-added products or to develop sustainable approaches for environmental remediation. The ATP-binding cassette proteins (ABCs) of Chlamydomonas reinhardtii have been characterized as indispensable transporters for CO2 concentrating mechanism, lipid biosynthesis, and heavy metal sequestration. However, few microalgal ABC proteins have been studied compared with higher plants or non-photosynthetic microorganisms. This study performed a genome-wide, evolutionary, and transcriptomic survey of C. reinhardtii ABC proteins (CrABCs). A total of 75 CrABCs were identified and classed into eight ABC subfamilies, from ABCA to ABCI. We found that no whole or partial genome duplication events occurred in C. reinhardtii after the ancient endosymbiosis events, but gene duplications occurred in a small range of chromosomal regions, which forced ABC family expansion. Abundant light, abscisic acid, and jasmonic acid response cis-elements were mapped in the CrABC promoters, coinciding with the evolutionary history of hormone signaling in Chlorophyta. The expression survey under light/dark rhythms revealed a close bond of CrABCs with cell division and development. A broad study of CrABCs supported their expected roles in heavy metal detoxification, lipid metabolism, and environmental adaptation. Moreover, the evolutionary and expression survey predicted the functions of unknown CrABCs, which are elaborated in the text. Two half-size CrABCGs-CrABCG3 and CrABCG26-were described as plasma-membrane transporters that might participate in lipidic compound secretion. This study provides fundamental and exhaustive information about CrABCs, which are indispensable for the functional elucidation of ABC proteins in microalgae.

Effect of arbuscular mycorrhizal fungi (Glomus mosseae) and elevated air temperature on Cd migration in the rhizosphere soil of alfalfa.

Cadmium (Cd) migration in the rhizosphere soil is easily affected by plants and microorganisms. Global warming significantly affects plant growth, and arbuscular mycorrhizal fungi (AMF) can chelate heavy metals by mycelium, cell wall components, and mycelial secretion. Here, we investigated the regulation of Glomus mosseae on Cd migration in the rhizosphere soil of alfalfa under elevated temperature (ET, + 3 °C). Elevated temperature significantly decreased G. mosseae colonization rate in the roots by 49.5% under Cd exposure. Under ET + G. mosseae + Cd relative to ET + Cd, the contents of free amino acids, total and easily extractable glomalin-related soil protein (GRSP), and root Cd increased significantly; however, the changes in DTPA-Cd in the rhizosphere soil and Cd in the shoots were insignificant. In addition, G. mosseae colonization enhanced the bioconcentration factor of Cd in the roots and the total removal rate of Cd in the rhizosphere soil by 63.4% and 16.3%, respectively, under ET + Cd. However, the changes in the expression of iron-regulated transport 1 (IRT1) and natural resistance-associated macrophage protein 1 genes were insignificant under ET + G. mosseae + Cd relative to ET + Cd. In summary, temperature and G. mosseae significantly affected Cd fate in the rhizosphere soil, and IRT1 gene and rhizosphere soil pH, N, and C/N ratio were significant factors influencing Cd migration. Additionally, G. mosseae improved the remediation efficiency of Cd-contaminated soils by alfalfa under ET. The results will help us understand the regulation of AMF on the phytoremediation of heavy metal-contaminated soils under global warming scenarios.

The Oncopig as an Emerging Model to Investigate Copper Regulation in Cancer.

Emerging evidence points to several fundamental contributions that copper (Cu) has to promote the development of human pathologies such as cancer. These recent and increasing identification of the roles of Cu in cancer biology highlights a promising field in the development of novel strategies against cancer. Cu and its network of regulatory proteins are involved in many different contextual aspects of cancer from driving cell signaling, modulating cell cycle progression, establishing the epithelial-mesenchymal transition, and promoting tumor growth and metastasis. Human cancer research in general requires refined models to bridge the gap between basic science research and meaningful clinical trials. Classic studies in cultured cancer cell lines and animal models such as mice and rats often present caveats when extended to humans due to inherent genetic and physiological differences. However, larger animal models such as pigs are emerging as more appropriate tools for translational research as they present more similarities with humans in terms of genetics, anatomical structures, organ sizes, and pathological manifestations of diseases like cancer. These similarities make porcine models well-suited for addressing long standing questions in cancer biology as well as in the arena of novel drug and therapeutic development against human cancers. With the emergent roles of Cu in human health and pathology, the pig presents an emerging and valuable model to further investigate the contributions of this metal to human cancers. The Oncopig Cancer Model is a transgenic swine model that recapitulates human cancer through development of site and cell specific tumors. In this review, we briefly outline the relationship between Cu and cancer, and how the novel Oncopig Cancer Model may be used to provide a better understanding of the mechanisms and causal relationships between Cu and molecular targets involved in cancer.

Bile acid composition regulates the manganese transporter Slc30a10 in intestine.

Bile acids (BAs) comprise heterogenous amphipathic cholesterol-derived molecules that carry out physicochemical and signaling functions. A major site of BA action is the terminal ileum, where enterocytes actively reuptake BAs and express high levels of BA-sensitive nuclear receptors. BA pool size and composition are affected by changes in metabolic health, and vice versa. One of several factors that differentiate BAs is the presence of a hydroxyl group on C12 of the steroid ring. 12α-Hydroxylated BAs (12HBAs) are altered in multiple disease settings, but the consequences of 12HBA abundance are incompletely understood. We employed mouse primary ileum organoids to investigate the transcriptional effects of varying 12HBA abundance in BA pools. We identified Slc30a10 as one of the top genes differentially induced by BA pools with varying 12HBA abundance. SLC30A10 is a manganese efflux transporter critical for whole-body manganese excretion. We found that BA pools, especially those low in 12HBAs, induce cellular manganese efflux and that Slc30a10 induction by BA pools is driven primarily by lithocholic acid signaling via the vitamin D receptor. Administration of lithocholic acid or a vitamin D receptor agonist resulted in increased Slc30a10 expression in mouse ileum epithelia. These data demonstrate a previously unknown role for BAs in intestinal control of manganese homeostasis.

Low-molecular-weight ligands in plants: role in metal homeostasis and hyperaccumulation.

Mineral nutrition is one of the key factors determining plant productivity. In plants, metal homeostasis is achieved through the functioning of a complex system governing metal uptake, translocation, distribution, and sequestration, leading to the maintenance of a regulated delivery of micronutrients to metal-requiring processes as well as detoxification of excess or non-essential metals. Low-molecular-weight ligands, such as nicotianamine, histidine, phytochelatins, phytosiderophores, and organic acids, play an important role in metal transport and detoxification in plants. Nicotianamine and histidine are also involved in metal hyperaccumulation, which determines the ability of some plant species to accumulate a large amount of metals in their shoots. In this review we extensively summarize and discuss the current knowledge of the main pathways for the biosynthesis of these ligands, their involvement in metal uptake, radial and long-distance transport, as well as metal influx, isolation and sequestration in plant tissues and cell compartments. It is analyzed how diverse endogenous ligand levels in plants can determine their different tolerance to metal toxic effects. This review focuses on recent advances in understanding the physiological role of these compounds in metal homeostasis, which is an essential task of modern ionomics and plant physiology. It is of key importance in studying the influence of metal deficiency or excess on various physiological processes, which is a prerequisite to the improvement of micronutrient uptake efficiency and crop productivity and to the development of a variety of applications in phytoremediation, phytomining, biofortification, and nutritional crop safety.

Hepcidin-Ferroportin Interaction Controls Systemic Iron Homeostasis.

Despite its abundance in the environment, iron is poorly bioavailable and subject to strict conservation and internal recycling by most organisms. In vertebrates, the stability of iron concentration in plasma and extracellular fluid, and the total body iron content are maintained by the interaction of the iron-regulatory peptide hormone hepcidin with its receptor and cellular iron exporter ferroportin (SLC40a1). Ferroportin exports iron from duodenal enterocytes that absorb dietary iron, from iron-recycling macrophages in the spleen and the liver, and from iron-storing hepatocytes. Hepcidin blocks iron export through ferroportin, causing hypoferremia. During iron deficiency or after hemorrhage, hepcidin decreases to allow iron delivery to plasma through ferroportin, thus promoting compensatory erythropoiesis. As a host defense mediator, hepcidin increases in response to infection and inflammation, blocking iron delivery through ferroportin to blood plasma, thus limiting iron availability to invading microbes. Genetic diseases that decrease hepcidin synthesis or disrupt hepcidin binding to ferroportin cause the iron overload disorder hereditary hemochromatosis. The opposite phenotype, iron restriction or iron deficiency, can result from genetic or inflammatory overproduction of hepcidin.

Identification of Three Small Molecules That Can Selectively Influence Cellular Manganese Levels in a Mouse Striatal Cell Model.

Manganese (Mn) is a biologically essential metal, critical as a cofactor for numerous enzymes such a glutamine synthetase and kinases such as ataxia-telangiectasia mutated (ATM). Similar to other essential metals such as iron and zinc, proper levels of Mn need to be achieved while simultaneously being careful to avoid excess levels of Mn that can be neurotoxic. A lifetime of occupational exposure to Mn can often lead to a Parkinsonian condition, also known as "manganism", characterized by impaired gait, muscle spasms, and tremors. Despite the importance of its regulation, the mechanisms underlying the transport and homeostasis of Mn are poorly understood. Rather than taking a protein or gene-targeted approach, our lab recently took a high-throughput-screening approach to identify 41 small molecules that could significantly increase or decrease intracellular Mn in a neuronal cell model. Here, we report characterization of these small molecules, which we refer to as the "Mn toolbox". We adapted a Fura-2-based assay for measuring Mn concentration and for measuring relative concentrations of other divalent metals: nickel, copper, cobalt, and zinc. Of these 41 small molecules, we report here the identification of three that selectively influence cellular Mn but do not influence the other divalent metals tested. The patterns of activity across divalent metals and the discovery of Mn-selective small molecules has potential pharmacological and scientific utility.

Copper chaperone blocks amyloid formation via ternary complex.

Protein misfolding in cells is avoided by a network of protein chaperones that detect misfolded or partially folded species. When proteins escape these control systems, misfolding may result in protein aggregation and amyloid formation. We here show that aggregation of the amyloidogenic protein α-synuclein (αS), the key player in Parkinson's disease, is controlled by the copper transport protein Atox1 in vitro. Copper ions are not freely available in the cellular environment, but when provided by Atox1, the resulting copper-dependent ternary complex blocks αS aggregation. Because the same inhibition was found for a truncated version of αS, lacking the C-terminal part, it appears that Atox1 interacts with the N-terminal copper site in αS. Metal-dependent chaperoning may be yet another manner in which cells control its proteome.

The Medicago truncatula Yellow Stripe1-Like3 gene is involved in vascular delivery of transition metals to root nodules.

Symbiotic nitrogen fixation carried out in legume root nodules requires transition metals. These nutrients are delivered by the host plant to the endosymbiotic nitrogen-fixing bacteria living within the nodule cells, a process in which vascular transport is essential. As members of the Yellow Stripe-Like (YSL) family of metal transporters are involved in root to shoot transport, they should also be required for root to nodule metal delivery. The genome of the model legume Medicago truncatula encodes eight YSL proteins, four of them with a high degree of similarity to Arabidopsis thaliana YSLs involved in long-distance metal trafficking. Among them, MtYSL3 is a plasma membrane protein expressed by vascular cells in roots and nodules and by cortical nodule cells. Reducing the expression level of this gene had no major effect on plant physiology when assimilable nitrogen was provided in the nutrient solution. However, nodule functioning was severely impaired, with a significant reduction of nitrogen fixation capabilities. Further, iron and zinc accumulation and distribution changed. Iron was retained in the apical region of the nodule, while zinc became strongly accumulated in the nodule veins in the ysl3 mutant. These data suggest a role for MtYSL3 in vascular delivery of iron and zinc to symbiotic nitrogen fixation.

Biosynthesis of isonitrile lipopeptides by conserved nonribosomal peptide synthetase gene clusters in Actinobacteria.

A putative lipopeptide biosynthetic gene cluster is conserved in many species of Actinobacteria, including Mycobacterium tuberculosis and M. marinum, but the specific function of the encoding proteins has been elusive. Using both in vivo heterologous reconstitution and in vitro biochemical analyses, we have revealed that the five encoding biosynthetic enzymes are capable of synthesizing a family of isonitrile lipopeptides (INLPs) through a thio-template mechanism. The biosynthesis features the generation of isonitrile from a single precursor Gly promoted by a thioesterase and a nonheme iron(II)-dependent oxidase homolog and the acylation of both amino groups of Lys by the same isonitrile acyl chain facilitated by a single condensation domain of a nonribosomal peptide synthetase. In addition, the deletion of INLP biosynthetic genes in M. marinum has decreased the intracellular metal concentration, suggesting the role of this biosynthetic gene cluster in metal transport.

Phosphatidylinositol 3-phosphate-binding protein AtPH1 controls the localization of the metal transporter NRAMP1 in Arabidopsis.

"Too much of a good thing" perfectly describes the dilemma that living organisms face with metals. The tight control of metal homeostasis in cells depends on the trafficking of metal transporters between membranes of different compartments. However, the mechanisms regulating the location of transport proteins are still largely unknown. Developing Arabidopsis thaliana seedlings require the natural resistance-associated macrophage proteins (NRAMP3 and NRAMP4) transporters to remobilize iron from seed vacuolar stores and thereby acquire photosynthetic competence. Here, we report that mutations in the pleckstrin homology (PH) domain-containing protein AtPH1 rescue the iron-deficient phenotype of nramp3nramp4 Our results indicate that AtPH1 binds phosphatidylinositol 3-phosphate (PI3P) in vivo and acts in the late endosome compartment. We further show that loss of AtPH1 function leads to the mislocalization of the metal uptake transporter NRAMP1 to the vacuole, providing a rationale for the reversion of nramp3nramp4 phenotypes. This work identifies a PH domain protein as a regulator of plant metal transporter localization, providing evidence that PH domain proteins may be effectors of PI3P for protein sorting.