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

2000; Springer Nature; Volume: 19; Issue: 14 Linguagem: Inglês

10.1093/emboj/19.14.3704

1460-2075

Amanda Bird, Hui Zhao, Huan Luo, Laran T. Jensen, Chandra Srinivasan, Marguerite V. Evans‐Galea, Dennis R. Winge, David Eide,

Advanced biosensing and bioanalysis techniques

Article17 July 2000free access A dual role for zinc fingers in both DNA binding and zinc sensing by the Zap1 transcriptional activator Amanda J. Bird Amanda J. Bird Department of Nutritional Sciences, University of Missouri–Columbia, Columbia, MO, 65211 USA Search for more papers by this author Hui Zhao Hui Zhao Department of Nutritional Sciences, University of Missouri–Columbia, Columbia, MO, 65211 USA Search for more papers by this author Huan Luo Huan Luo Department of Nutritional Sciences, University of Missouri–Columbia, Columbia, MO, 65211 USA Search for more papers by this author Laran T. Jensen Laran T. Jensen Department of Biochemistry, University of Utah Health Sciences Center, Salt Lake City, UT, 84132 USA Search for more papers by this author Chandra Srinivasan Chandra Srinivasan Department of Biochemistry, University of Utah Health Sciences Center, Salt Lake City, UT, 84132 USA Search for more papers by this author Marguerite Evans-Galea Marguerite Evans-Galea Department of Biochemistry, University of Utah Health Sciences Center, Salt Lake City, UT, 84132 USA Search for more papers by this author Dennis R. Winge Dennis R. Winge Department of Biochemistry, University of Utah Health Sciences Center, Salt Lake City, UT, 84132 USA Search for more papers by this author David J. Eide Corresponding Author David J. Eide Department of Nutritional Sciences, University of Missouri–Columbia, Columbia, MO, 65211 USA Search for more papers by this author Amanda J. Bird Amanda J. Bird Department of Nutritional Sciences, University of Missouri–Columbia, Columbia, MO, 65211 USA Search for more papers by this author Hui Zhao Hui Zhao Department of Nutritional Sciences, University of Missouri–Columbia, Columbia, MO, 65211 USA Search for more papers by this author Huan Luo Huan Luo Department of Nutritional Sciences, University of Missouri–Columbia, Columbia, MO, 65211 USA Search for more papers by this author Laran T. Jensen Laran T. Jensen Department of Biochemistry, University of Utah Health Sciences Center, Salt Lake City, UT, 84132 USA Search for more papers by this author Chandra Srinivasan Chandra Srinivasan Department of Biochemistry, University of Utah Health Sciences Center, Salt Lake City, UT, 84132 USA Search for more papers by this author Marguerite Evans-Galea Marguerite Evans-Galea Department of Biochemistry, University of Utah Health Sciences Center, Salt Lake City, UT, 84132 USA Search for more papers by this author Dennis R. Winge Dennis R. Winge Department of Biochemistry, University of Utah Health Sciences Center, Salt Lake City, UT, 84132 USA Search for more papers by this author David J. Eide Corresponding Author David J. Eide Department of Nutritional Sciences, University of Missouri–Columbia, Columbia, MO, 65211 USA Search for more papers by this author Author Information Amanda J. Bird1, Hui Zhao1, Huan Luo1, Laran T. Jensen2, Chandra Srinivasan2, Marguerite Evans-Galea2, Dennis R. Winge2 and David J. Eide 1 1Department of Nutritional Sciences, University of Missouri–Columbia, Columbia, MO, 65211 USA 2Department of Biochemistry, University of Utah Health Sciences Center, Salt Lake City, UT, 84132 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:3704-3713https://doi.org/10.1093/emboj/19.14.3704 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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 C2H2 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. Introduction Metal ions such as iron, copper and zinc are essential nutrients but can also be cytotoxic if accumulated in excess amounts. Therefore, in the face of ever-changing extracellular or dietary levels, organisms must maintain adequate intracellular supplies of these metals for cellular metabolism while preventing their overaccumulation. In general, under metal-limiting conditions, pathways are upregulated to allow efficient scavenging of the metals from the extracellular environment or utilization of intracellular stores. Under conditions of metal excess, other systems are induced that facilitate metal ion efflux from the cell or promote intracellular sequestration in membrane-bound organelles or binding to macromolecules such as metallothionein, phytochelatin and ferritin. Regardless of the specific homeostatic regulatory mechanism in question, intracellular metallosensing proteins play key roles in controlling these processes. For example, MTF-1 is a zinc sensor protein in vertebrates that upregulates the transcription of metallothionein genes in response to excess zinc levels (Heuchel et al., 1994). The iron sensor proteins IRP1 and IRP2 control both the translation of ferritin mRNA as well as the stability of the transferrin receptor mRNA to regulate simultaneously iron storage and uptake (Eisenstein and Blemings, 1998). These are just two examples of a growing number of metallosensing regulatory proteins that have been identified in studies at all phylogenetic levels. A common theme emerging from these many studies is that metallosensors monitor intracellular metal ion status by direct binding of the metal (or metal-containing complexes, e.g. heme) to regulatory sites within the protein that then modulate the activity of the metallosensor (O'Halloran, 1993). Thus, an understanding of the precise mechanism that these regulatory proteins use to sense metal ion levels is clearly essential for understanding of metal ion homeostasis. Our recent studies have focused on the mechanisms of zinc homeostasis in the yeast Saccharomyces cerevisiae. In this yeast, zinc uptake is mediated by the Zrt1 and Zrt2 zinc transporters found in the plasma membrane (Zhao and Eide, 1996a,b; Gitan et al., 1998). The activity of these transporters is controlled by two separate zinc-responsive mechanisms. First, their activity is regulated at a post-translational level by controlling the rate of their removal from the cell surface in response to zinc status (Gitan et al., 1998; Gitan and Eide, 2000). Treating cells with high concentrations of zinc triggers endocytosis of the zinc transporters and this regulation prevents overaccumulation of the metal. The zinc-responsive metallosensor(s) controlling this process has not been identified. The second zinc-responsive regulatory mechanism in yeast occurs at the transcriptional level. Both the ZRT1 and ZRT2 genes are expressed at high levels in zinc-limited cells and their expression is shut off at high zinc levels (Zhao and Eide, 1996a,b). The Zap1 transcriptional activator is directly responsible for this regulation (Zhao and Eide, 1997). In addition to the zinc transporter genes, Zap1 upregulates expression of its own promoter via a positive autoregulatory mechanism. This autoregulation was proposed to increase the magnitude of the transcriptional response to zinc deficiency (Zhao and Eide, 1997). Zap1 also controls the export of stored zinc from the vacuole by regulating expression of Zrt3, a putative vacuolar zinc efflux transporter (MacDiarmid et al., 2000). In addition to these genes, DNA microarray analysis suggested that Zap1 controls the expression of as many as 42 other genes in response to zinc status (Lyons et al., 2000). Clearly, this protein is a major component in the regulation of cellular zinc homeostasis. Zap1 is a 93 kDa protein with seven potential zinc finger domains. Five of these domains are clustered at the C-terminus of the protein and constitute the intact DNA-binding domain (Zhao et al., 1998; Bird et al., 2000). These five fingers are required for high-affinity and sequence-specific DNA binding to sites, called zinc-responsive elements (ZREs), which are found in the promoters of Zap1 target genes. The current consensus ZRE sequence, derived from mutational studies as well as comparison of many such elements from potential Zap1 target gene promoters, is 5′-ACCTTNAAGGT-3′ (Zhao et al., 1998; Lyons et al., 2000). Zap1 also contains two regions of high acidic residue content that were previously proposed to be activation domains (Zhao and Eide, 1997). Two key questions regarding Zap1's role as a zinc metallosensory protein are (i) what is the mechanism of zinc sensing used by this regulatory system and (ii) how does the zinc signal control Zap1 function? We show here that Zap1 activity is regulated by zinc largely at a post-translational level. To address the mechanism of how Zap1 senses zinc, we have mapped the zinc-responsive domain (ZRD) of the protein to the five zinc fingers also required for DNA binding. Furthermore, our data demonstrate that at least some of these fingers, in addition to their role in DNA binding, are required for the zinc-responsive regulation of Zap1 activity. These results suggest a novel role for zinc fingers in both metal sensing and DNA binding. Results Regulation of Zap1 activity by zinc occurs at a post-translational level In wild-type cells where Zap1 is expressed from its own promoter, expression of a ZRE-lacZ fusion is induced ∼90-fold in zinc-limited cells relative to cells grown under zinc-replete conditions (Figure 1A). In previous studies, we demonstrated that Zap1 activates its own expression during zinc deficiency, which presumably amplifies the transcriptional response. To determine what contribution this autoregulation makes to the overall zinc responsiveness of Zap1p-regulated gene expression, we fused the ZAP1 protein coding region to the GAL1 promoter and examined regulation in a zap1 mutant strain (Figure 1A). Expression of the GAL1 promoter was previously found to be unaffected by zinc availability (Lyons et al., 2000). Six myc epitope tags were also introduced at the N-terminus of GAL1-expressed Zap1 to facilitate the detection and localization of the protein (see below). This epitope-tagged protein (myc-Zap1) fully complemented the growth defect of a zap1 mutant strain in low zinc, indicating that the fusion retained wild-type function (data not shown). The GAL1 promoter vector alone conferred no ZRE-lacZ expression in either low or high zinc. When the myc-Zap1 protein was expressed from this promoter at a high level in galactose-grown cells, they showed ∼8-fold induction of ZRE-lacZ expression in low zinc, i.e. only 10% of the wild-type regulation. This apparently lower level of regulation is largely due to increased basal lacZ expression in the cells expressing high levels of Zap1. Low level expression of myc-Zap1 from the GAL1 promoter in cells grown on glucose, a carbon source that represses GAL1 expression 1000-fold (St John and Davis, 1981; Johnston et al., 1994), was still sufficient to fully complement the zap1 mutation (data not shown) and confer nearly wild-type zinc-regulated expression (∼50-fold). Thus, while autoregulation may contribute slightly to overall zinc responsiveness, most of the regulation occurs through post-transcriptional control of Zap1p activity. Figure 1.Regulation of Zap1 activity by zinc occurs at a post-translational level. (A) Wild-type (DY1457) and zap1 mutant (ZHY6) cells containing either the pYef2 vector or pMyc-Zap11–880 were grown to exponential phase in LZM-galactose supplemented with either 5 μM (−Zn) or 1000 μM (+Zn) ZnCl2. Zap1 activity in each strain was assessed using the pDg2 ZRE-lacZ reporter. ZHY6 pMyc-Zap11–880 transformants were also assayed after growth in glucose, a carbon source that represses most but not all expression from the GAL1 promoter. A representative experiment is shown and the error bars indicate 1 SD. (B) The stability of Zap1 is not affected by zinc status. Wild-type (DY1457) cells transformed with the pYef2 vector and zap1 mutant (ZHY6) cells bearing pMyc-Zap11–880 were grown in LZM-galactose to exponential phase. The concentrations of ZnCl2 added to the medium were 5 (lane 2), 250 (lane 3), 500 (lane 4) and 1000 μM (lanes 1 and 5). Crude protein extracts were prepared, fractionated by SDS–PAGE analysis, and assayed for Zap1 and Vph1 protein levels by immunoblotting. Download figure Download PowerPoint Post-transcriptional control of Zap1 could occur through zinc-regulated changes in translation efficiency or protein stability. To test these hypotheses, we used immunoblotting to determine the effects of differing zinc availability on Zap1 level (Figure 1B). An immunoblot of the Vph1 vacuolar ATPase subunit showed equal protein loading in all lanes. In a vector-transformed zap1 mutant, no Zap1 protein was observed in protein extracts when probed with the anti-myc antibody. Several bands, ranging in size from ∼45 to 120 kDa, were detected in protein extracts of cells expressing myc-Zap1. The predicted molecular mass of myc-Zap1 is 105 kDa and two bands were observed that were of this approximate size. We noted that Zap1 was particularly sensitive to proteolysis during preparation of these samples (data not shown), so smaller forms may result from partial degradation of the protein. Alternatively, the highest molecular mass form may result from protein modification (e.g. phosphorylation) of full-length Zap1. Whatever their source(s), all forms of Zap1 detected were present in similar relative amounts across a broad range of zinc concentrations, indicating that none represented forms of the protein associated with its post-transcriptional control by zinc. Thus, we concluded that Zap1 zinc responsiveness occurs at a post-translational level. Zap1 is present in the nucleus of zinc-deficient and zinc-replete cells Many transcription factors are regulated post-translationally by controlling their nuclear localization. To determine whether zinc similarly affects Zap1 protein trafficking, we used indirect immunofluorescence to observe myc-Zap1's subcellular location in zinc-deficient and -replete cells (Figure 2). Little fluorescence was observed in vector-transformed cells. Myc-Zap1 fluorescence was present in cells expressing the tagged allele and grown under both deficient and replete conditions. This fluorescence co-localized with the DNA-staining reagent 4′,6′-diamidino-2-phenylindole dihydrochloride (DAPI), demonstrating that Zap1 was localized to the nucleus in both low and high zinc. These results show that regulation of Zap1 by zinc does not occur through altered protein trafficking. Figure 2.The subcellular localization of Zap1 is not affected by zinc status. Wild-type (DY1457) cells bearing the vector pYef2 and zap1 mutant (ZHY6) cells bearing pMyc-Zap11–880 were grown to exponential phase in LZM-galactose supplemented with either 5 μM (−Zn) or 1000 μM (+Zn) ZnCl2. Cells were viewed by Nomarski optics or epifluorescence. DAPI was used to stain the nucleus and the myc-Zap1 protein was detected by indirect immunofluorescence. The blue fluorescence of DAPI staining was converted to red and the DAPI and myc-Zap1 images were overlaid using Adobe Photoshop (merge). Yellow color in the merged images indicates colocalization of the markers. Download figure Download PowerPoint Mapping the ZRD of Zap1 with Gal4 DNA-binding domain fusions By a process of elimination, we concluded from the data shown in Figures 1 and 2 that the activity of Zap1 is regulated through the control of its DNA-binding and/or activation domain function. To gain insight into these possible mechanisms of regulation, we conducted a functional dissection of Zap1 using a 'one-hybrid' protein fusion approach. The main goal of this analysis was to identify the ZRD of Zap1. The primary amino acid sequence of Zap1 suggested a number of domains that are common to transcriptional activators (Figure 3A). These include a C-terminal region that contains seven putative zinc finger domains (Zhao and Eide, 1997) and two regions rich in acidic residues (glutamate and aspartate) that could potentially be transcriptional activation domains. We have recently demonstrated that the five C-terminal zinc fingers of Zap1 are both necessary and sufficient for ZRE binding activity in vivo and in vitro (Zhao et al., 1998; Bird et al., 2000). Figure 3.The functional domains of Zap1. (A) Schematic representation of Zap1. Shown are the functional activation domains (AD1 and AD2, hatched boxes) and the DNA-binding domain. The seven putative zinc finger domains are represented by black boxes and are numbered 1–7. Amino acid positions relevant to the constructed plasmid fusions are also numbered. The position of the Cys→Ser substitution in the Zap1-1up protein is shown. (B) Mapping the ZRD of Zap1. The activity of GBD–Zap1 fusion proteins expressed from the ADH1 promoter in the strain DEY1538 (zap1 gal4 gal80) was measured using either a ZRE-lacZ (pDg2) or a GAL1-lacZ (pRY171) reporter. Cells were grown in LZM supplemented with either 5 μM (−Zn) or 1000 μM (+Zn) ZnCl2 prior to the β-galactosidase activity assays. A representative experiment is shown and the error bars indicate 1 SD. Accumulation of all fusion proteins was confirmed by immunoblot analysis using an anti-GBD antibody. EMSA with a ZRE oligonucleotide probe (Zhao et al., 1998) was used to assess DNA-binding activity. ND, not determined. Download figure Download PowerPoint To identify the ZRD of Zap1, we first fused the Gal4 DNA-binding domain (GBD) to the N-terminus of either full-length Zap1 or various Zap1 truncates to generate a series of fusion proteins designated GBD–Zap1x−y (where x and y denote the endpoints of the Zap1 region included; deletion endpoints are denoted by Δx–y) (Figure 3B). Because the GBD can bind to its own upstream activation sequence, the activity of these potentially bifunctional fusion proteins could be assayed using either a GAL1-lacZ reporter or a ZRE-lacZ reporter. As controls, immunoblot analysis using an anti-GBD antibody was used to ensure that the fusion proteins were produced and electrophoretic mobility shift assays (EMSA) were used to determine which fusion proteins retained ZRE binding activity. Nuclear localization of inactive fusions was also confirmed by indirect immunofluorescence. The GBD fusion proteins were expressed from the ADH1 promoter in a zap1 gal4 mutant strain (DEY1538). Transformation of this strain with the GBD vector alone failed to confer expression on either ZRE-lacZ or GAL1-lacZ reporters (Figure 3B). The full-length GBD–Zap1 fusion (GBD–Zap11–880) fully complemented the growth defect of the zap1 mutation in low zinc (data not shown) and resulted in significant zinc-responsive gene expression on the ZRE-lacZ reporter. Given that this fusion is expressed from the ADH1 promoter, whose expression is also not upregulated in low zinc (Lyons et al., 2000), these results support our other experiments, indicating that autoregulation of the ZAP1 promoter plays a minor role in the overall regulation. The results also demonstrated that the GBD fusion does not interfere with the repression of Zap1 activity by this high zinc condition (see below). When activity of GBD–Zap11–880 was tested on the GAL1-lacZ reporter, a similarly high degree of zinc regulation was also observed. Thus, Zap1 can confer regulation on a heterologous DNA-binding domain fused to its N-terminus. This ability indicated that we could use this approach to map the Zap1 ZRD independently of its DNA-binding activity. Given that our previous studies indicated that the first two zinc fingers of Zap1, ZnF1 and ZnF2, were not required for DNA binding, an attractive hypothesis was that these two fingers function in zinc-responsive regulation (Bird et al., 2000). However, this was clearly not the case; deletion of both ZnF1 and ZnF2 (GBD–Zap1Δ553–686) retained wild-type zinc-responsive gene expression on both ZRE-lacZ and GAL1-lacZ reporters. The N-terminal deletions GBD–Zap1342–880 (data not shown), GBD–Zap1552–880 and GBD–Zap1611–880 also showed wild-type levels of zinc-responsive expression on both reporters, indicating that each retained fully functional ZRD activity. GBD–Zap1642–880 had no activity on either promoter despite accumulating to high levels in the nucleus and having ZRE-binding activity. This suggested that residues 611–642 contained activation domain function (see below). Truncations were also generated from the C-terminal end; GBD–Zap1552–850, in which the last 29 amino acids of Zap1 were removed, showed wild-type zinc regulation. Therefore, this region, which contains an eighth potential zinc finger with the non-canonical sequence of C-X2-C-X12-Q-X3-C, is not required for either DNA binding or zinc responsiveness. Deletion of ZnF7 (GBD–Zap1552–825) resulted in loss of ZRE-lacZ expression, consistent with this finger being required for DNA binding (Bird et al., 2000). Remarkably, this fusion had nearly constitutive expression on the GAL1-lacZ reporter. Taken together, these results mapped the ZRD of Zap1 to amino acids 642–850. Furthermore, they demonstrated that residues 825–851, which contain ZnF7, are required for both DNA binding and zinc-responsive regulation of Zap1. GBD fusions were also useful in mapping the activation domains of Zap1. GBD–Zap1552–705 conferred expression on the GAL1-lacZ reporter. These data, together with other results described in Figure 3B, indicated that one Zap1 activation domain, designated as AD2, is located between amino acids 611 and 642. Expression of the GAL1-lacZ reporter by GBD–Zap1552–705 was actually higher in zinc-replete cells than in zinc-limited cells, probably due to the negative effects of zinc deficiency on overall gene expression that we have observed previously (Zhao and Eide, 1996a). This conclusion was further supported by the observation that a GBD fusion to the p53 activation domain showed similarly increased activity in high zinc (GBD–p53AD). That additional activation domain function is found elsewhere in Zap1 was suggested by the activity of GBD–Zap1Δ553–686, which lacks AD2. Consistent with this hypothesis, GBD–Zap11–552, which also lacks AD2, conferred expression on the GAL1-lacZ reporter (Figure 3C). Neither GBD–Zap11–331 nor GBD–Zap11–187 showed GAL1-lacZ expression despite being stable, nuclear (data not shown) proteins. These results demonstrated that another activation domain, designated AD1, is located between amino acids 331 and 552. As expected, none of the fusions lacking the Zap1 DNA-binding domain showed ZRE binding in vitro or expression from the ZRE-lacZ reporter in vivo. The experiments described in Figure 3 are consistent with a single ZRD in Zap1 localizing to residues 642–851. An alternative hypothesis is that there are multiple independent ZRDs in the protein that, because of their differences in sensitivity to zinc, might allow for regulation of gene expression over a broader range of concentrations than a single domain could provide. Subtle differences in the zinc responsiveness of different fusion proteins that this model predicts could be missed when only examining their activities at very high and very low zinc concentrations. To determine whether the different GBD fusions respond differently to zinc, we assayed ZRE-lacZ expression over a range of zinc concentrations. On the ZRE-lacZ reporter, these fusions were similarly repressed at the highest concentration of zinc used (Figures 3B and 4A). However, marked differences were observed at intermediate zinc concentrations (Figure 4A). Activity of GBD–Zap1552–880 was repressed at lower zinc concentrations than GBD–Zap11–880. Furthermore, even higher levels of zinc were required to repress the activity of GBD–Zap1Δ553–686. The differential regulation of these various fusions in response to zinc initially suggested that more than one ZRD might be present in Zap1. Further analysis demonstrated that this was not the case and that the differential effects were an artefact of the GBD fusions. When these same Zap1 proteins were analyzed as myc fusions, all were found to have identical zinc dose– response curves (Figure 4B). Thus, a single zinc-responsive mechanism is at work in these various proteins. These data also independently support the GBD mapping of the ZRD to the C-terminus of Zap1; i.e. the only region common to all of these similarly regulated fusions is residues 687–880. Figure 4.Comparison of GBD–Zap1 and myc-Zap1 activities in response to a range of zinc concentrations. (A) A zap1 mutant strain (ZHY6) bearing the pDg2 ZRE-lacZ reporter and pGBD–Zap11–880 (filled squares), pGBD–Zap1Δ553–686 (filled diamonds) or pGBD–Zap1552–880 (open circles) was grown to exponential phase in LZM supplemented with the indicated concentrations of ZnCl2 prior to assay for β-galactosidase activity. (B) The same zap1 mutant strain bearing the pDg2 ZRE-lacZ reporter and pMyc-Zap11–880 (filled squares), pMyc-Zap1Δ553–686 (filled diamonds) or pMyc-Zap1552–880 (open circles) was grown to exponential phase in LZM supplemented with the indicated concentrations of ZnCl2 prior to assay for β-galactosidase activity. Shown are representative experiments in which the standard deviations were <10% of the corresponding mean. Download figure Download PowerPoint Mapping the ZRD of Zap1 with Gal4 activation domain fusions Mapping of the Zap1 ZRD to its DNA-binding domain suggested the usefulness of a complementary approach to examine this regulation, i.e. via activation domain fusions. This method potentially allows mapping of the ZRD independently of Zap1's activation domain function. The Gal4 activation domain (GAD) was fused to the N-terminus of full-length Zap1 and Zap1 truncates, and examined for regulation of a ZRE-lacZ reporter in response to zinc. Because the Gal80 protein inhibits GAD function in glucose-grown cells, these fusions were analyzed in a zap1 gal4 gal80 mutant. Furthermore, the proteins were expressed from the GAL1 promoter in this strain through action of the β-estradiol-responsive GEV activator protein, a fusion of the GBD, the human estrogen receptor hormone response domain and the herpes virus VP16 activation domain (C.Y.Gao and J.L.Pinkham, manuscript submitted). This expression system has the additional advantage of controlling the levels of protein expression by increasing doses of the inducer, β-estradiol. At low levels of expression (β-estradiol = 10−8 M), the GAD–Zap1–880 fusion complemented a zap1 mutation (data not shown) and retained highly zinc-responsive regulation (Figure 5A). This indicated that the GAD did not interfere with the normal regulation of Zap1 when expressed at a low level. Furthermore, the results demonstrated that the ZRD of Zap1 can confer this regulation not only on a heterologous DBD but also on a foreign activation domain. Fusions of various Zap1 truncates to the GAD confirmed mapping of the ZRD to the same region of the protein required for DNA binding, i.e. amino acids 687–880. All fusions conferred strong zinc-responsive regulation of expression including the smallest fusion, GAD–Zap1687–880. These results confirm the mapping of the ZRD to this region of the protein. Figure 5.Mapping the ZRD with GAD fusions. The indicated fusions were expressed in a zap1 gal4 gal80 mutant strain (DEY1538) bearing the pDg2 ZRE-lacZ reporter and pGEV-HIS3. pGEV-HIS3 encodes a hybrid transcriptional activator that is induced by β-estradiol. (A) Cells were grown to exponential phase in LZM supplemented with either 5 μM (−Zn) or 1000 μM (+Zn) ZnCl2 and either 10−8 or 10−6 M β-estradiol. β-galactosidase activities are represented as a percentage of the corresponding −Zn expression level and the standard deviations were <10% of the corresponding mean. (B) The same transformants as in (A) were grown to exponential phase in LZM supplemented with 1000 μM ZnCl2; protein extracts were prepared and analyzed by immunoblotting using anti-GAD or anti-Vph1 antibodies. Apparent proteolytic products were observed in some samples; i.e. the largest band in each lane corresponds to the expected molecular mass of each fusion. Download figure Download PowerPoint At higher levels of expression (i.e. β-estradiol = 10−6 M), some of the fusions (e.g. GAD–Zap1552–880, GAD–Zap1642–880 and GAD–Zap1687–880) no longer showed zinc responsiveness. While the precise cause of this effect is not known (see Discussion), immunoblots demonstrated that these particular fusion proteins accumulated to higher levels than those forms that retained zinc regulation when their mRNAs were overexpressed (Figure 5B). Thus, as is true for other transcription factors, studies of Zap1 require careful control of expression levels to obtain meaningful results. DNA-binding zinc finger domains are structural determinants of the ZRD The ZRD of Zap1 contains the five zinc fingers ZnF3–ZnF7 (Figure 3A), each of which we have shown previ