Two of the five zinc fingers in the Zap1 transcription factor DNA binding domain dominate site-specific DNA binding.

The Zap1 transcriptional activator from Saccharomyces cerevisiae induces expression of a series of genes containing an 11 base pair conserved promoter element (ZRE) under conditions of zinc deficiency. This work shows that Zap1 uses four of its seven zinc finger domains to contact the ZRE and that two of these dominate the interaction by contacting the essential ACC-GGT ends. Two Zn finger domains (ZF1 and ZF2) do not contact DNA, and a third ZF3 may be more important for interfinger protein-protein interactions. Zn finger domains important for ZRE contact were identified from triple mutations in Zap1, changing three residues in the alpha helix in each finger known to be important for DNA contacts in Zn finger proteins. Replacement of -1, 3, and 6 helix residues in ZF4 and ZF7 reduced the affinity of Zap1 for the wild-type ZRE. In contrast, triple mutations within the intervening ZF5 and ZF6 domains had minimal effect. The data argue that fingers 4 and 7 contact the ACC-GGT ends while fingers 5 and 6 contact the 5 bp central ZRE sequence. This conclusion is corroborated by decreased Zap1 affinity for a ZRE DNA duplex containing mutations of the AC-GT ends of the ZRE, whereas transversion mutations within the central 5 bp of the ZRE had minimal effect on Zap1 binding affinity.

Functional genomics and metal metabolism.

Metal ions are essential nutrients, yet they can also be toxic if they over-accumulate. Homeostatic mechanisms and detoxification systems therefore precisely control their intracellular levels and distribution. The tools of functional genomics are rapidly accelerating understanding in this field, particularly in the yeast Saccharomyces cerevisiae.

The human ZIP1 transporter mediates zinc uptake in human K562 erythroleukemia cells.

The ZIP superfamily of transporters plays important roles in metal ion uptake in diverse organisms. There are 12 ZIP-encoding genes in humans, and we hypothesize that many of these proteins are zinc transporters. In this study, we addressed the role of one human ZIP gene, hZIP1, in zinc transport. First, we examined (65)Zn uptake activity in K562 erythroleukemia cells overexpressing hZIP1. These cells accumulated more zinc than control cells because of increased zinc influx. Moreover, consistent with its role in zinc uptake, hZIP1 protein was localized to the plasma membrane. Our results also demonstrated that hZIP1 is responsible for the endogenous zinc uptake activity in K562 cells. hZIP1 is expressed in untransfected K562 cells, and the increase in mRNA levels found in hZIP1-overexpressing cells correlated with the increased zinc uptake activity. Furthermore, hZIP1-dependent (65)Zn uptake was biochemically indistinguishable from the endogenous activity. Finally, inhibition of endogenous hZIP1 expression with antisense oligonucleotides caused a marked decrease in endogenous (65)Zn uptake activity. The observation that hZIP1 is the major zinc transporter in K562 cells, coupled with its expression in many normal cell types, indicates that hZIP1 plays an important role in zinc uptake in human tissues.

Eukaryotic zinc transporters and their regulation.

The last ten years have witnessed major advances in our understanding of zinc transporters and their regulation in eukaryotic organisms. Two families of transporters, the ZIP (Zrt-, Irt-like Protein) and CDF (Cation Diffusion Facilitator) families, have been found to play a number of important roles in zinc transport. These are ancient gene families that span all phylogenetic levels. The characterized members of each group have been implicated in the transport of metal ions, frequently zinc, across lipid bilayer membranes. This remarkable conservation of function suggests that other, as yet uncharacterized members of the family, will also be involved in metal ion transport. Many of the ZIP family transporters are involved in cellular zinc uptake and at least one member, the Zrt3 transporter of S. cerevisiae, transports stored zinc out of an intracellular compartment during adaptation to zinc deficiency. In contrast, CDF family members mediate zinc efflux out of cells or facilitate zinc transport into intracellular compartments for detoxification and/or storage. The activity of many of these transporters is regulated in response to zinc through transcriptional and post-transcriptional mechanisms to maintain zinc homeostasis at both the cellular and organismal levels.

The Fe(II) permease Fet4p functions as a low affinity copper transporter and supports normal copper trafficking in Saccharomyces cerevisiae.

The plasma-membrane of Saccharomyces cerevisiae contains high affinity permeases for Cu(I) and Fe(II). A low affinity Fe(II) permease has also been identified, designated Fet4p. A corresponding low affinity copper permease has not been characterized, although yeast cells that lack high affinity copper uptake do accumulate this metal ion. We demonstrate in the present study that Fet4p can function as a low affinity copper permease. Copper is a non-competitive inhibitor of (55)Fe uptake through Fet4p (K(i)=22 microM). Fet4p-dependent (67)Cu uptake was kinetically characterized, with K(m) and V(max) values of 35 microM and 8 pmol of copper/min per 10(6) cells respectively. A fet4-containing strain exhibited no saturable, low affinity copper uptake indicating that this uptake was attributable to Fet4p. Mutant forms of Fet4p that exhibited decreased efficiency in (55/59)Fe uptake were similarly compromised in (67)Cu uptake, indicating that similar amino acid residues in Fet4p contribute to both uptake processes. The copper taken into the cell by Fet4p was metabolized similarly to the copper taken into the cell by the high affinity permease, Ctr1p. This was shown by the Fet4p-dependence of copper activation of Fet3p, the copper oxidase that supports high affinity iron uptake in yeast. Also, copper-transported by Fet4p down-regulated the copper sensitive transcription factor, Mac1p. Whether supplied by Ctr1p or by Fet4p, an intracellular copper concentration of approx. 10 microM caused a 50% reduction in the transcriptional activity of Mac1p. The data suggest that the initial trafficking of newly arrived copper in the yeast cell is independent of the copper uptake pathway involved, and that this copper may be targeted first to a presumably small 'holding' pool prior to its partitioning within the cell.

Altered selectivity in an Arabidopsis metal transporter.

Plants require metals for essential functions ranging from respiration to photosynthesis. These metals also contribute to the nutritional value of plants for both humans and livestock. Additionally, plants have the ability to accumulate nonessential metals such as cadmium and lead, and this ability could be harnessed to remove pollutant metals from the environment. Designing a transporter that specifically accumulates certain cations while excluding others has exciting applications in all of these areas. The Arabidopsis root membrane protein IRT1 is likely to be responsible for uptake of iron from the soil. Like other Fe(II) transporters identified to date, IRT1 transports a variety of other cations, including the essential metals zinc and manganese as well as the toxic metal cadmium. By heterologous expression in yeast, we show here that the replacement of a glutamic acid residue at position 103 in wild-type IRT1 with alanine increases the substrate specificity of the transporter by selectively eliminating its ability to transport zinc. Two other mutations, replacing the aspartic acid residues at either positions 100 or 136 with alanine, also increase IRT1 metal selectivity by eliminating transport of both iron and manganese. A number of other conserved residues in or near transmembrane domains appear to be essential for all transport function. Therefore, this study identifies at least some of the residues important for substrate selection and transport in a protein belonging to the ZIP gene family, a large transporter family found in a wide variety of organisms.

A dual role for zinc fingers in both DNA binding and zinc sensing by the Zap1 transcriptional activator.

The Zap1 transcriptional activator of Saccharomyces cerevisiae controls zinc homeostasis. Zap1 induces target gene expression in zinc-limited cells and is repressed by high zinc. One such target gene is ZAP1 itself. In this report, we examine how zinc regulates Zap1 function. First, we show that transcriptional autoregulation of Zap1 is a minor component of zinc responsiveness; most regulation of Zap1 activity occurs post-translationally. Secondly, nuclear localization of Zap1 does not change in response to zinc, suggesting that zinc regulates DNA binding and/or activation domain function. To understand how Zap1 responds to zinc, we performed a functional dissection of the protein. Zap1 contains two activation domains. DNA-binding activity is conferred by five C-terminal C(2)H(2) zinc fingers and each finger is required for high-affinity DNA binding. The zinc-responsive domain of Zap1 also maps to the C-terminal zinc fingers. Furthermore, mutations that disrupt some of these fingers cause constitutive activity of a bifunctional Gal4 DNA-binding domain-Zap1 fusion protein. These results demonstrate a novel function of Zap1 zinc fingers in zinc sensing as well as DNA binding.

Metal ion transport in eukaryotic microorganisms: insights from Saccharomyces cerevisiae.

Metal ions such as iron, copper, manganese, and zinc are essential nutrients for all eukaryotic microorganisms. Therefore, these organisms possess efficient uptake mechanisms to obtain these nutrients from their extracellular environment. Metal ions must also be transported into intracellular organelles where they function as catalytic and structural cofactors for compartmentalized enzymes. Thus, intracellular transport mechanisms are also present. When present in high levels, metal ions can also be toxic, so their uptake and intracellular transport is tightly regulated at both transcriptional and post-transcriptional levels to limit metal ion overaccumulation and facilitate storage and sequestration. Remarkable molecular insight into these processes has come from recent studies of the yeast Saccharomyces cerevisiae. This organism, which is the primary subject of this chapter, serves as a useful paradigm to understand metal ion metabolism in other eukaryotic microbes.

Genome-wide characterization of the Zap1p zinc-responsive regulon in yeast.

The Zap1p transcription factor senses cellular zinc status and increases expression of its target genes in response to zinc deficiency. Previously known Zap1p-regulated genes encode the Zrt1p, Zrt2p, and Zrt3p zinc transporter genes and Zap1p itself. To allow the characterization of additional genes in yeast important for zinc homeostasis, a systematic study of gene expression on the genome-wide scale was used to identify other Zap1p target genes. Using a combination of DNA microarrays and a computer-assisted analysis of shared motifs in the promoters of similarly regulated genes, we identified 46 genes that are potentially regulated by Zap1p. Zap1p-regulated expression of seven of these newly identified target genes was confirmed independently by using lacZ reporter fusions, suggesting that many of the remaining candidate genes are also Zap1p targets. Our studies demonstrate the efficacy of this combined approach to define the regulon of a specific eukaryotic transcription factor.

Mapping the DNA binding domain of the Zap1 zinc-responsive transcriptional activator.

The Zap1 transcriptional activator of Saccharomyces cerevisiae plays a major role in zinc homeostasis by inducing the expression of several genes under zinc-limited growth conditions. This activation of gene expression is mediated by binding of the protein to one or more zinc-responsive elements present in the promoters of its target genes. To better understand how Zap1 functions, we mapped its DNA binding domain using a combined in vivo and in vitro approach. Our results show that the Zap1 DNA binding domain maps to the carboxyl-terminal 194 amino acids of the protein; this region contains five of its seven potential zinc finger domains. Fusing this region to the Gal4 activation domain complemented a zap1Delta mutation for low zinc growth and also conferred high level expression on a zinc-responsive element-lacZ reporter. In vitro, the purified 194-residue fragment bound to DNA with a high affinity (dissociation constant in the low nanomolar range) similar to that of longer fragments of Zap1. Furthermore, by deletion and site-directed mutagenesis, we demonstrated that each of the five carboxyl-terminal zinc fingers are required for high affinity DNA binding.

Zinc-regulated ubiquitin conjugation signals endocytosis of the yeast ZRT1 zinc transporter.

The yeast ZRT1 zinc transporter is regulated by zinc at both transcriptional and post-translational levels. At the post-translational level, zinc inactivates ZRT1 by inducing the removal of the protein from the plasma membrane by endocytosis. The zinc transporter is subsequently degraded in the vacuole. This regulatory system allows for the rapid shut off of zinc uptake activity in cells exposed to high zinc concentrations, thereby preventing overaccumulation of this potentially toxic metal. In this report, we examine the role of ubiquitin conjugation in this process. First, we show that ZRT1 is ubiquitinated shortly after zinc treatment and before endocytosis. Secondly, mutations in various components of the ubiquitin conjugation pathway, specifically the RSP5 ubiquitin-protein ligase and the UBC4 and UBC5 ubiquitin conjugating enzymes, inhibit both ubiquitination and endocytosis. Finally, mutation of a specific lysine residue in ZRT1 blocks both ubiquitination and endocytosis. This critical lysine, Lys-195, is located in a cytoplasmic loop region of the protein and may be the residue to which ubiquitin is attached. These results demonstrate that ubiquitin conjugation is a critical step in the signal transduction pathway that controls the rate of ZRT1 endocytosis in response to zinc.

Functional expression of the human hZIP2 zinc transporter.

Zinc is an essential nutrient for humans, yet we know little about how this metal ion is taken up by mammalian cells. In this report, we describe the characterization of hZip2, a human zinc transporter identified by its similarity to zinc transporters recently characterized in fungi and plants. hZip2 is a member of the ZIP family of eukaryotic metal ion transporters that includes two other human genes, hZIP1 and hZIP3, and genes in mice and rats. To test whether hZip2 is a zinc transporter, we examined (65)Zn uptake activity in transfected K562 erythroleukemia cells expressing hZip2 from the CMV promoter. hZip2-expressing cells accumulated more zinc than control cells because of an increased initial zinc uptake rate. This activity was time-, temperature-, and concentration-dependent and saturable with an apparent K(m) of 3 microM. hZip2 zinc uptake activity was inhibited by several other transition metals, suggesting that this protein may transport other substrates as well. hZip2 activity was not energy-dependent, nor did it require K(+) or Na(+) gradients. Zinc uptake by hZip2 was stimulated by HCO(3)(-) treatment, suggesting a Zn(2+)-HCO(3)(-) cotransport mechanism. Finally, hZip2 was exclusively localized in the plasma membrane. These results indicate that hZip2 is a zinc transporter, and its identification provides one of the first molecular tools to study zinc uptake in mammalian cells.

The molecular biology of metal ion transport in Saccharomyces cerevisiae.

Transition metals such as iron, copper, manganese, and zinc are essential nutrients. The yeast Saccharomyces cerevisiae is an ideal organism for deciphering the mechanism and regulation of metal ion transport. Recent studies of yeast have shown that accumulation of any single metal ion is mediated by two or more substrate-specific transport systems. High-affinity systems are active in metal-limited cells, whereas low-affinity systems play the predominant roles when the substrate is more abundant. Metal ion uptake systems of cells are tightly controlled, and both transcriptional and posttranscriptional regulatory mechanisms have been identified. Most importantly, studies of S. cerevisiae have identified a large number of genes that function in metal ion transport and have illuminated the existence of importance of gene families that play related roles in these processes in mammals.

TAT1 encodes a low-affinity histidine transporter in Saccharomyces cerevisiae.

Previous studies have revealed the presence of at least two histidine uptake systems in S. cerevisiae; one with high affinity and the other with low affinity for histidine. The HIP1 gene is known to encode the high affinity permease. The purpose of this study was to identify the gene that encodes the low affinity permease. A mutant strain of S. cerevisiae that is both a histidine auxotroph and a hip1 deletion mutant is unable to grow on low histidine media. This strain was transformed with a yeast cDNA library constructed in a yeast expression vector. Transformants with increased histidine transport were selected by their ability to grow on a low histidine media. Sequencing of the inserts revealed the presence of the HIP1 gene and also the presence of the TAT1 gene. Estimated Km and Vmax values for histidine transport by each system were determined. In a hip1 tat1 double mutant, the level of histidine required for growth increased eight-fold in comparison to the hip1 single mutant. Our results suggest that the TAT1-encoded protein, previously characterized as the high-affinity tyrosine permease, also acts as the low affinity histidine permease.

Zap1p, a metalloregulatory protein involved in zinc-responsive transcriptional regulation in Saccharomyces cerevisiae.

Zinc ion homeostasis in Saccharomyces cerevisiae is controlled primarily through the transcriptional regulation of zinc uptake systems in response to intracellular zinc levels. A high-affinity uptake system is encoded by the ZRT1 gene, and its expression is induced more than 30-fold in zinc-limited cells. A low-affinity transporter is encoded by the ZRT2 gene, and this system is also regulated by zinc. We used a genetic approach to isolate mutants whose ZRT1 expression is no longer repressed in zinc-replete cells, and a new gene, ZAP1, was identified. ZAP1 encodes a 93-kDa protein with sequence similarity to transcriptional activators; the C-terminal 174 amino acids contains five C2H2 zinc finger domains, and the N terminus (residues 1 to 706) has two potential acidic activation domains. The N-terminal region also contains 12% histidine and cysteine residues. The mutant allele isolated, ZAP1-1up, is semidominant and caused high-level expression of ZRT1 and ZRT2 in both zinc-limited and zinc-replete cells. This phenotype is the result of a mutation that substitutes a serine for a cysteine residue in the N-terminal region. A zap1 deletion mutant grew well on zinc-replete media but poorly on zinc-limiting media. This mutant had low-level ZRT1 and ZRT2 expression in zinc-limited as well as zinc-replete cells. These data indicate that Zap1p plays a central role in zinc ion homeostasis by regulating transcription of the zinc uptake system genes in response to zinc. Finally, we present evidence that Zap1p regulates transcription of its own promoter in response to zinc through a positive autoregulatory mechanism.

The FET4 gene encodes the low affinity Fe(II) transport protein of Saccharomyces cerevisiae.

Previous studies on Fe(II) uptake in Saccharomyces cerevisiae suggested the presence of two uptake systems with different affinities for this substrate. We demonstrate that the FET3 gene is required for high affinity uptake but not for the low affinity system. This requirement has enabled a characterization of the low affinity system. Low affinity uptake is time-, temperature-, and concentration-dependent and prefers Fe(II) over Fe(III) as substrate. We have isolated a new gene, FET4, that is required for low affinity uptake, and our results suggest that FET4 encodes an Fe(II) transporter protein. FET4's predicted amino acid sequence contains six potential transmembrane domains. Overexpressing FET4 increased low affinity uptake, whereas disrupting this gene eliminated that activity. In contrast, overexpressing FET4 decreased high affinity activity, while disrupting FET4 increased that activity. Therefore, the high affinity system may be regulated to compensate for alterations in low affinity activity. These analyses, and the analysis of the iron-dependent regulation of the plasma membrane Fe(III) reductase, demonstrate that the low affinity system is a biologically relevant mechanism of iron uptake in yeast. Furthermore, our results indicate that the high and low affinity systems are separate uptake pathways.

The GEF1 gene of Saccharomyces cerevisiae encodes an integral membrane protein; mutations in which have effects on respiration and iron-limited growth.

We have isolated a new class of respiration-defective, i.e petite, mutants of the yeast Saccharomyces cerevisiae. Mutations in the GEF1 gene cause cells to grow slowly on rich media containing carbon sources utilized by respiration. This phenotype is suppressed by adding high concentrations of iron to the growth medium. Gef1- mutants also fail to grow on a fermentable carbon source, glucose, when iron is reduced to low concentrations in the medium, suggesting that the GEF1 gene is required for efficient metabolism of iron during growth on fermentable as well as respired carbon sources. However, activity of the iron uptake system appears to be unaffected in gef1- mutants. Fe(II) transporter activity and regulation is normal in gef1- mutants. Fe(III) reductase induction during iron-limited growth is disrupted, but this appears to be a secondary effect of growth rate alterations. The wild-type GEF1 gene was cloned and sequenced; it encodes a protein of 779 amino acids, 13 possible transmembrane domains, and significant similarity to chloride channel proteins from fish and mammals, suggesting that GEF1 encodes an integral membrane protein. A gef1- deletion mutation generated in vitro and introduced into wild-type haploid strains by gene transplacement was not lethal. Oxygen consumption by intact gef1- cells and by mitochondrial fractions isolated from gef1- mutants was reduced 25-50% relative to wild type, indicating that mitochondrial function is defective in these mutants. We suggest that GEF1 encodes a transport protein that is involved in intracellular iron metabolism.

The vacuolar H(+)-ATPase of Saccharomyces cerevisiae is required for efficient copper detoxification, mitochondrial function, and iron metabolism.

Mutations in the GEF2 gene of the yeast Saccharomyces cerevisiae have pleiotropic effects. The gef2 mutants display a petite phenotype. These cells grow slowly on several different carbon sources utilized exclusively or primarily by respiration. This phenotype is suppressed by adding large amounts of iron to the growth medium. A defect in mitochondrial function may be the cause of the petite phenotype: the rate of oxygen consumption by intact gef2 cells and by mitochondrial fractions isolated from gef2 mutants was reduced 60%-75% relative to wild type. Cytochrome levels were unaffected in gef2 mutants, indicating that heme accumulation is not significantly altered in these strains. The gef2 mutants were also more sensitive than wild type to growth inhibition by several divalent cations including Cu. We found that the cup5 mutation, causing Cu sensitivity, is allelic to gef2 mutations. The GEF2 gene was isolated, sequenced, and found to be identical to VMA3, the gene encoding the vacuolar H(+)-ATPase proteolipid subunit. These genetic and biochemical analyses demonstrate that the vacuolar H(+)-ATPase plays a previously unknown role in Cu detoxification, mitochondrial function, and iron metabolism.

Novel insertion mutation in Caenorhabditis elegans.

The mutation e1662 is an allele of the Caenorhabditis elegans unc-54 gene induced with the difunctional alkylating agent 1,2,7,8-diepoxyoctane. unc-54 encodes the major myosin heavy chain isozyme of body wall muscle cells. Filter-transfer hybridization and DNA sequence analysis show that e1662 is an insertion of 288 base pairs of DNA within unc-54. The inserted DNA is identical to a 288-base pair region of unc-54 located ca. 600 base pairs from the insertion site. Thus, e1662 is a displaced duplication. A 14-base pair sequence located at one end of the duplicated segment is found adjacent to the site of insertion. These homologous sequences are juxtaposed head-to-tail by the insertion event. e1662 thus contains a tandem direct repeat extending across one of its junctions.