Biomedical subjects

D Eide

Publications and source records attributed to D Eide.

At least 19 recordsLinked to original sources

PubMed2000

Zinc transporters that regulate vacuolar zinc storage in Saccharomyces cerevisiae.

All cells regulate their intracellular zinc levels. In yeast, zinc uptake is mediated by Zrt1p and Zrt2p, which belong to the ZIP family of metal transporters. Under zinc limitation, ZRT1 and ZRT2 transcription is induced by the Zap1p transcriptional activator. We describe here a new component of zinc homeostasis, vacuolar zinc storage, that is also regulated by Zap1p. Zinc-replete cells accumulate zinc in the vacuole via the Zrc1p and Cot1p transporters. Our results indicate that another zinc transporter, Zrt3p, mobilizes this stored zinc in zinc-limited cells. ZRT3 is a Zap1p-regulated gene whose transcription increases in low zinc. Zrt3p is also a member of the ZIP family and it localizes to the vacuolar membrane. The effects of ZRT3 mutation and overexpression on cell growth, cellular zinc accumulation and intracellular labile zinc pools are all consistent with its proposed role. Furthermore, we demonstrate that zrt3 mutants inefficiently mobilize stored zinc to offset deficiency. Thus, our studies define a system of zinc influx and efflux transporters in the vacuole that play important roles in zinc homeostasis.

Amino Acid Sequence↗

PubMed2000

The molecular physiology of heavy metal transport in the Zn/Cd hyperaccumulator Thlaspi caerulescens.

An integrated molecular and physiological investigation of the fundamental mechanisms of heavy metal accumulation was conducted in Thlaspi caerulescens, a Zn/Cd-hyperaccumulating plant species. A heavy metal transporter cDNA, ZNT1, was cloned from T. caerulescens through functional complementation in yeast and was shown to mediate high-affinity Zn(2+) uptake as well as low-affinity Cd(2+) uptake. It was found that this transporter is expressed at very high levels in roots and shoots of the hyperaccumulator. A study of ZNT1 expression and high-affinity Zn(2+) uptake in roots of T. caerulescens and in a related nonaccumulator, Thlaspi arvense, showed that alteration in the regulation of ZNT1 gene expression by plant Zn status results in the overexpression of this transporter and in increased Zn influx in roots of the hyperaccumulating Thlaspi species. These findings yield insights into the molecular regulation and control of plant heavy metal and micronutrient accumulation and homeostasis, as well as provide information that will contribute to the advancement of phytoremediation by the future engineering of plants with improved heavy metal uptake and tolerance.

Amino Acid Sequence↗

PubMed1999

Zeroing in on zinc uptake in yeast and plants.

Zinc is an essential micronutrient. Genes responsible for zinc uptake have now been identified from yeast and plants. These genes belong to an extended family of cation transporters called the ZIP gene family. Zinc efflux genes that belong to another transporter family, the CDF family, have also been identified in yeast and Arabidopsis. It is clear that studies in yeast can greatly aid our understanding of zinc metabolism in plants.

Animals↗

PubMed1999

The IRT1 protein from Arabidopsis thaliana is a metal transporter with a broad substrate range.

The molecular basis for the transport of manganese across membranes in plant cells is poorly understood. We have found that IRT1, an Arabidopsis thaliana metal ion transporter, can complement a mutant Saccharomyces cerevisiae strain defective in high-affinity manganese uptake (smf1 delta). The IRT1 protein has previously been identified as an iron transporter. The current studies demonstrated that IRT1, when expressed in yeast, can transport manganese as well. This manganese uptake activity was inhibited by cadmium, iron(II) and zinc, suggesting that IRT1 can transport these metals. The IRT1 cDNA also complements a zinc uptake-deficient yeast mutant strain (zrt1zrt2), and IRT1-dependent zinc transport in yeast cells is inhibited by cadmium, copper, cobalt and iron(III). However, IRT1 did not complement a copper uptake-deficient yeast mutant (ctr1), implying that this transporter is not involved in the uptake of copper in plant cells. The expression of IRT1 is enhanced in A. thaliana plants grown under iron deficiency. Under these conditions, there were increased levels of root-associated manganese, zinc and cobalt, suggesting that, in addition to iron, IRT1 mediates uptake of these metals into plant cells. Taken together, these data indicate that the IRT1 protein is a broad-range metal ion transporter in plants.

Arabidopsis↗

PubMed1998

Sequence analyses and phylogenetic characterization of the ZIP family of metal ion transport proteins.

Several novel but similar heavy metal ion transporters, Zrt1, Zrt2, Zip1-4 and Irt1, have recently been characterized. Zrt1, Zrt2 and Zip1-4 are probably zinc transporters in Saccharomyces cerevisiae and Arabidopsis thaliana whereas Irt1 appears to play a role in iron uptake in A. thaliana. The family of proteins including these functionally characterized transporters has been designated the Zrt- and Irt-related protein (ZIP) family. In this report, ZIP family proteins in the current databases were identified and multiply aligned, and a phylogenetic tree for the family was constructed. A family specific signature sequence was derived, and the available sequences were analyzed for residues of potential functional significance. A fully conserved intramembranous histidyl residue, present within a putative amphipathic, alpha-helical, transmembrane spanning segment, was identified which may serve as a part of an intrachannel heavy metal ion binding site. The occurrence of a proposed extramembranal metal binding motif (H X H X H) was examined in order to evaluate its potential functional significance for various members of the family. The computational analyses reported in this topical review should serve as a guide to future researchers interested in the structure-function relationships of ZIP family proteins.

Amino Acid Sequence↗

PubMed1998

Zinc-induced inactivation of the yeast ZRT1 zinc transporter occurs through endocytosis and vacuolar degradation.

The ZRT1 gene encodes the transporter responsible for high affinity zinc uptake in yeast. ZRT1 is transcribed in zinc-limited cells and its transcription is repressed in zinc-replete cells. In this report, we describe a second, post-translational mechanism that regulates ZRT1 activity. In zinc-limited cells, ZRT1 is a stable, N-glycosylated plasma membrane protein. Exposure to high levels of extracellular zinc triggers a rapid loss of ZRT1 uptake activity. Our results demonstrate that this inactivation occurs through zinc-induced endocytosis of the protein and its subsequent degradation in the vacuole. Mutations that inhibit the internalization step of endocytosis also inhibited zinc-induced ZRT1 inactivation and the major vacuolar proteases were required to degrade ZRT1 in response to zinc. Furthermore, immunofluorescence microscopy showed that ZRT1 is localized to the plasma membrane in zinc-limited cells and that the protein is transferred to the vacuole via an endosome-like compartment upon exposure to zinc. ZRT1 inactivation is a relatively specific response to zinc; cadmium and cobalt ions trigger the response but less effectively than zinc. Moreover, zinc does not alter the stability of several other plasma membrane proteins. Therefore, zinc-induced ZRT1 inactivation is a specific regulatory system to shut off zinc uptake activity in cells exposed to high extracellular zinc levels thereby preventing overaccumulation of this potentially toxic metal.

Carrier Proteins↗

PubMed1998

Regulation of zinc homeostasis in yeast by binding of the ZAP1 transcriptional activator to zinc-responsive promoter elements.

Zinc homeostasis in yeast is controlled primarily through the regulation of zinc uptake. Transcription of the ZRT1 and ZRT2 zinc transporters increases in zinc-limited cells, and this induction is dependent on the ZAP1 gene. We hypothesized previously that ZAP1 encodes a zinc-responsive transcriptional activator. Expression of ZAP1 itself increases in zinc-limited cells. This response is also dependent on ZAP1 function through a potential positive autoregulatory mechanism. In this report, we describe the characterization of zinc-responsive elements (ZREs) in the promoters of the ZRT1, ZRT2, and ZAP1 genes. A ZRE consensus sequence, 5'-ACCYYNAAGGT-3', was identified and found to be both necessary and sufficient for zinc-responsive transcriptional regulation. We also demonstrate that ZREs are DNA binding sites for ZAP1. First, a dominant ZAP1 mutation, ZAP1-1(up), which causes increased expression of ZAP1-regulated genes in zinc-replete cells, exerted its effects specifically through the ZREs. Second, electrophoretic mobility shift assays and in vitro DNase I footprint analyses indicated that ZAP1 binds to ZREs in a sequence-specific fashion. These studies demonstrate that ZAP1 plays a direct role in controlling zinc-responsive gene expression in yeast by binding to zinc-responsive elements in the promoters of genes that it regulates.

Alleles↗

PubMed1998

Identification of a family of zinc transporter genes from Arabidopsis that respond to zinc deficiency.

Millions of people worldwide suffer from nutritional imbalances of essential metals like zinc. These same metals, along with pollutants like cadmium and lead, contaminate soils at many sites around the world. In addition to posing a threat to human health, these metals can poison plants, livestock, and wildlife. Deciphering how metals are absorbed, transported, and incorporated as protein cofactors may help solve both of these problems. For example, edible plants could be engineered to serve as better dietary sources of metal nutrients, and other plant species could be tailored to remove metal ions from contaminated soils. We report here the cloning of the first zinc transporter genes from plants, the ZIP1, ZIP2, and ZIP3 genes of Arabidopsis thaliana. Expression in yeast of these closely related genes confers zinc uptake activities. In the plant, ZIP1 and ZIP3 are expressed in roots in response to zinc deficiency, suggesting that they transport zinc from the soil into the plant. Although expression of ZIP2 has not been detected, a fourth related Arabidopsis gene identified by genome sequencing, ZIP4, is induced in both shoots and roots of zinc-limited plants. Thus, ZIP4 may transport zinc intracellularly or between plant tissues. These ZIP proteins define a family of metal ion transporters that are found in plants, protozoa, fungi, invertebrates, and vertebrates, making it now possible to address questions of metal ion accumulation and homeostasis in diverse organisms.

Amino Acid Sequence↗

PubMed1997

Characterization of the FET4 protein of yeast. Evidence for a direct role in the transport of iron.

The low affinity Fe2+ uptake system of Saccharomyces cerevisiae requires the FET4 gene. In this report, we present evidence that FET4 encodes the Fe2+ transporter protein of this system. Antibodies prepared against FET4 detected two distinct proteins with molecular masses of 63 and 68 kDa. In vitro synthesis of FET4 suggested that the 68-kDa form is the primary translation product, and the 63-kDa form may be generated by proteolytic cleavage of the full-length protein. Consistent with its role as an Fe2+ transporter, FET4 is an integral membrane protein present in the plasma membrane. The level of FET4 closely correlated with uptake activity over a broad range of expression levels and is itself regulated by iron. Furthermore, mutations in FET4 can alter the kinetic properties of the low affinity uptake system, suggesting a direct interaction between FET4 and its Fe2+ substrate. Mutations affecting potential Fe2+ ligands located in the predicted transmembrane domains of FET4 significantly altered the apparent Km and/or Vmax of the low affinity system. These mutations may identify residues involved in Fe2+ binding during transport.

Biological Transport↗

PubMed1997

The yeast FET5 gene encodes a FET3-related multicopper oxidase implicated in iron transport.

The yeast FET3 gene encodes an integral membrane multicopper oxidase required for high-affinity iron uptake. The FET4 gene encodes an Fe(II) transporter required for low-affinity uptake. To identify other yeast genes involved in iron uptake, we isolated genes that could, when overexpressed, suppress the iron-limited growth defect of a fet3 fet4 mutant. The FET5 gene was isolated in this screen and it encodes a multi-copper oxidase closely related to Fet3p. Several observations indicate that Fet5p plays a role analogous to Fet3p in iron transport. Suppression of the fet3 fet4 mutant phenotype by FET5 overexpression required the putative FTR1 transporter subunit of the high-affinity system. Fet5p is an integral membrane protein whose oxidase domain is located on the cell surface or within an intracellular compartment. Oxidase activity measured in cells with altered levels of FET5 expression suggested that Fet5p is a functional oxidase. FET5 overexpression increased the rate of iron uptake by a novel uptake system. Finally, FET5 mRNA levels are regulated by iron and are increased in cells grown in iron-limited media. These results suggest that Fet5p normally plays a role in the transport of iron.

Biological Transport↗

PubMed1997

Molecular biology of iron and zinc uptake in eukaryotes.

Recent studies of iron uptake in Saccharomyces cerevisiae have provided insights into the role of multicopper oxidases in eukaryotic metal transport. These studies have also led to the identification of a novel iron transporter in plants and the recognition of a new family of transporter proteins that may participate in metal uptake in a diverse array of eukaryotic species.

Animals↗

PubMed1996

The ZRT2 gene encodes the low affinity zinc transporter in Saccharomyces cerevisiae.

Zinc accumulation in Saccharomyces cerevisiae occurs through either of two uptake systems. A high affinity system is active in zinc-limited cells, and the ZRT1 gene encodes the transporter protein of this system. In this study, we characterized the low affinity system that is active in zinc-replete cells. The low affinity system is time-, temperature-, and concentration-dependent and prefers zinc over other metals as its substrate. Our results suggest that the ZRT2 gene encodes the transporter of this system. The amino acid sequence of Zrt2p is remarkably similar to those of Zrt1p and Irt1p, an Fe2+ transporter from Arabidopsis thaliana. Overexpressing ZRT2 increased low affinity uptake, whereas disrupting this gene eliminated that activity, but had little effect on the high affinity system. Therefore, the high and low affinity systems are separate uptake pathways. Analysis of the zinc levels required for growth of zrt2 mutant strains as well as the effects of the zrt2 mutation on the regulation of the high affinity system demonstrated that the low affinity system is a biologically relevant mechanism of zinc accumulation. Finally, a zrt1zrt2 mutant was viable, indicating the existence of additional zinc uptake pathways.

Amino Acid Sequence↗

PubMed1996

A novel iron-regulated metal transporter from plants identified by functional expression in yeast.

Iron is an essential nutrient for virtually all organisms. The IRT1 (iron-regulated transporter) gene of the plant Arabidopsis thaliana, encoding a probable Fe(II) transporter, was cloned by functional expression in a yeast strain defective for iron uptake. Yeast expressing IRT1 possess a novel Fe(II) uptake activity that is strongly inhibited by Cd. IRT1 is predicted to be an integral membrane protein with a metal-binding domain. Data base comparisons and Southern blot analysis indicated that IRT1 is a member of a gene family in Arabidopsis. Related sequences were also found in the genomes of rice, yeast, nematodes, and humans. In Arabidopsis, IRT1 is expressed in roots, is induced by iron deficiency, and has altered regulation in plant lines bearing mutations that affect the iron uptake system. These results provide the first molecular insight into iron transport by plants.

Amino Acid Sequence↗

PubMed1996

The yeast ZRT1 gene encodes the zinc transporter protein of a high-affinity uptake system induced by zinc limitation.

The yeast Saccharomyces cerevisiae has two separate systems for zinc uptake. One system has high affinity for substrate and is induced in zinc-deficient cells. The second system has lower affinity and is not highly regulated by zinc status. The ZRT1 gene encodes the transporter for the high-affinity system, called Zrt1p. The predicted amino acid sequence of Zrt1p is similar to that of Irt1p, a probable Fe(II) transporter from Arabidopsis thaliana. Like Irt1p, Zrt1p contains eight potential transmembrane domains and a possible metal-binding domain. Consistent with the proposed role of ZRT1 in zinc uptake, overexpressing this gene increased high-affinity uptake activity, whereas disrupting it eliminated that activity and resulted in poor growth of the mutant in zinc-limited media. Furthermore, ZRT1 mRNA levels and uptake activity were closely correlated, as was zinc-limited induction of a ZRT1-lacZ fusion. These results suggest that ZRT1 is regulated at the transcriptional level by the intracellular concentration of zinc. ZRT1 is an additional member of a growing family of metal transport proteins.

Base Sequence↗

PubMed1995

The FET3 gene product required for high affinity iron transport in yeast is a cell surface ferroxidase.

The yeast FET3 gene is required for high affinity iron transport (Askwith, C., Eide, D., Ho, A. V., Bernard, P. S., Li, L., Davis-Kaplan, S., Sipe, D. M., and Kaplan, J. (1994) Cell 76, 403-410). The gene has extensive sequence homology to the family of multi-copper oxidases. In this communication, we demonstrate that the gene product is a cell surface ferroxidase involved in iron transport. Cells that contain a functional FET3 gene product exhibited an iron-dependent non-mitochondrial increase in oxygen consumption. Comparison of the rate of iron oxidation to O2 consumption yielded an approximate value of 4:1, as predicted for a ferroxidase. Spheroplasts obtained from cells grown under low iron conditions also displayed an iron-dependent increase in O2 consumption. Treatment of spheroplasts with trypsin or affinity-purified antibodies directed against the putative external ferroxidase domain of Fet3 had no effect on basal O2 consumption but inhibited the iron-dependent increase in O2 consumption. Anti-peptide antibodies directed against the cytosolic domain of Fet3 had no effect on O2 consumption. These studies indicate that Fet3 is a plasma membrane ferroxidase required for high affinity iron uptake, in which the ferroxidase-containing domain is localized on the external cell surface.

Amino Acid Sequence↗

PubMed1994

Molecular characterization of a copper transport protein in S. cerevisiae: an unexpected role for copper in iron transport.

We report the identification and characterization of CTR1, a gene in the yeast S. cerevisiae that encodes a multispanning plasma membrane protein specifically required for high affinity copper transport into the cell. The predicted protein contains a methionine- and serine-rich domain that includes 11 examples of the sequence Met-X2-Met, a motif noted in proteins involved in bacterial copper metabolism. CTR1 mutants and deletion strains have profound deficiency in ferrous iron uptake, thus revealing a requirement for copper in mediating ferrous transport into the cell. Genetic evidence suggests that the target for this requirement is the FET3 gene (detailed in a companion study), predicted to encode a copper-containing protein that acts as a cytosolic ferro-oxidase. These findings provide an unexpected mechanistic link between the uptake of copper and iron.

Amino Acid Sequence↗

PubMed1994

The FET3 gene of S. cerevisiae encodes a multicopper oxidase required for ferrous iron uptake.

S. cerevisiae accumulate iron by a process requiring a ferrireductase and a ferrous transporter. We have isolated a mutant, fet3, defective for high affinity Fe(II) uptake. The wild-type FET3 gene was isolated by complementation of the mutant defect. Sequence analysis of the gene revealed the presence of an open reading frame coding for a protein with strong similarity to the family of blue multicopper oxidoreductases. Consistent with the role of copper in iron transport, growth of wild-type cells in copper-deficient media resulted in decreased ferrous iron transport. Addition of copper, but not other transition metals (manganese or zinc), to the assay media resulted in the recovery of Fe(II) transporter activity. We suggest that the catalytic activity of the Fet3 protein is required for cellular iron accumulation.

Amino Acid Sequence↗

PubMed1992

Regulation of iron uptake in Saccharomyces cerevisiae. The ferrireductase and Fe(II) transporter are regulated independently.

Iron is required for the growth of Saccharomyces cerevisiae. High concentrations of iron, however, are toxic, forcing this yeast to tightly regulate its concentration of intracellular free iron. We demonstrate that S. cerevisiae accumulates iron through the combined action of a plasma membrane ferrireductase and an Fe(II) transporter. This transporter is highly selective for Fe(II). Several other transition metals did not inhibit iron uptake when these metals were present at a concentration 100-fold higher than the Km (0.15 microM) for iron transport. Pt(II) inhibited ferrireductase activity but not the ability of cells to transport iron that was chemically reduced to Fe(II). Incubation of cells in a synthetic iron-limited media resulted in the induction of both ferrireductase and Fe(II) transporter activities. In complex media, Fe(II) transport activity was regulated in response to media iron concentration, while the activity of the ferrireductase was not. When stationary phase cells were inoculated into fresh media, ferrireductase activity increased independent of the iron content of the media; in contrast, transporter activity varied inversely with iron levels. These results demonstrate that the ferrireductase and Fe(II) transporter are separately regulated and that iron accumulation may be limited by changes in either activity.

Biological Transport↗