Zinc fingers can act as Zn2+ sensors to regulate transcriptional activation domain function
2003; Springer Nature; Volume: 22; Issue: 19 Linguagem: Inglês
10.1093/emboj/cdg484
ISSN1460-2075
Autores Tópico(s)RNA and protein synthesis mechanisms
ResumoArticle1 October 2003free access Zinc fingers can act as Zn2+ sensors to regulate transcriptional activation domain function Amanda J. Bird Amanda J. Bird Department of Nutritional Sciences, 217 Gwynn Hall, University of Missouri, Columbia, MO, 65211 USA Search for more papers by this author Keith McCall Keith McCall Department of Medicine and Biochemistry, University of Utah Health Sciences Center, Salt Lake City, UT, 84132 USA Search for more papers by this author Michelle Kramer Michelle Kramer Department of Nutritional Sciences, 217 Gwynn Hall, University of Missouri, Columbia, MO, 65211 USA Search for more papers by this author Elizabeth Blankman Elizabeth Blankman Department of Medicine and 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 Medicine and 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, 217 Gwynn Hall, University of Missouri, Columbia, MO, 65211 USA Search for more papers by this author Amanda J. Bird Amanda J. Bird Department of Nutritional Sciences, 217 Gwynn Hall, University of Missouri, Columbia, MO, 65211 USA Search for more papers by this author Keith McCall Keith McCall Department of Medicine and Biochemistry, University of Utah Health Sciences Center, Salt Lake City, UT, 84132 USA Search for more papers by this author Michelle Kramer Michelle Kramer Department of Nutritional Sciences, 217 Gwynn Hall, University of Missouri, Columbia, MO, 65211 USA Search for more papers by this author Elizabeth Blankman Elizabeth Blankman Department of Medicine and 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 Medicine and 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, 217 Gwynn Hall, University of Missouri, Columbia, MO, 65211 USA Search for more papers by this author Author Information Amanda J. Bird1, Keith McCall2, Michelle Kramer1, Elizabeth Blankman2, Dennis R. Winge2 and David J. Eide 1 1Department of Nutritional Sciences, 217 Gwynn Hall, University of Missouri, Columbia, MO, 65211 USA 2Department of Medicine and Biochemistry, University of Utah Health Sciences Center, Salt Lake City, UT, 84132 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:5137-5146https://doi.org/10.1093/emboj/cdg484 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The yeast Zap1 transcription factor controls the expression of genes involved in zinc accumulation and storage. Zap1 is active in zinc-limited cells and repressed in replete cells. Zap1 has two activation domains, AD1 and AD2, which are both regulated by zinc. AD2 function was mapped to a region containing two Cys2His2 zinc fingers, ZF1 and ZF2, that are not involved in DNA binding. More detailed mapping placed AD2 almost precisely within the endpoints of ZF2, suggesting a role for these fingers in regulating activation domain function. Consistent with this hypothesis, ZF1 and ZF2 bound zinc in vitro but less stably than did zinc fingers involved in DNA binding. Furthermore, mutations predicted to disrupt zinc binding to ZF1 and/or ZF2 rendered AD2 constitutively active. Our results also indicate that the repressed form of AD2 requires an intramolecular interaction between ZF1 and ZF2. These studies suggest that these zinc fingers play an unprecedented role as zinc sensors to control activation domain function. Introduction The Cys2His2 (C2H2) zinc finger motif is ubiquitous in biology. This domain was first characterized in transcription factor IIIA (TFIIIA) by Klug and colleagues (Miller et al., 1985). Since then, hundreds of proteins containing these motifs have been identified. In the human genome alone, 3% of the ∼32 000 predicted open reading frames encode proteins with zinc fingers (Landers, 2001). Detailed characterization of a relatively small subset of these proteins has implicated zinc fingers in several functions. The most commonly recognized role of zinc fingers is in protein–DNA binding (Rhodes and Klug, 1993). Zinc fingers and related zinc-binding motifs have also been shown to act in the binding of proteins to RNA (Finerty and Bass, 1999), lipids (Gaullier et al., 1998) and other proteins (Mackay and Crossley, 1998). In this report, we present evidence for a novel role of these motifs as zinc sensors involved in regulating the activation domain of a transcription factor. Zinc is an essential nutrient but can be toxic to cells if accumulated in excess amounts. To survive, cells have mechanisms to maintain intracellular zinc homeostasis. The precision of zinc homeostasis was recently highlighted by studies of Escherichia coli, where intracellular zinc levels are controlled by the transcriptional regulation of both uptake and efflux transporters (Patzer and Hantke, 1998; Brocklehurst et al., 1999). Recent studies of the transcription factors responsible for this regulation suggest that these cells strive to maintain little or no free cytoplasmic zinc (Outten and O'Halloran, 2001). Several reports suggest that eukaryotic cells also maintain very low levels of cytoplasmic labile zinc (Sensi et al., 1997; Cheng and Reynolds, 1998). We know much about zinc homeostasis in eukaryotes through studies of the yeast Saccharomyces cerevisiae. In this yeast, zinc homeostasis is largely mediated by the regulation of uptake transporters and transporters involved in the intracellular storage of zinc in the vacuole. The Zrt1, Zrt2 and Fet4 proteins are metal ion transporters responsible for zinc uptake across the plasma membrane (Zhao and Eide, 1996a, b; Waters and Eide, 2002). Vacuolar zinc storage is controlled by the Zrc1 and Zrt3 transporters (MacDiarmid et al., 2000; Miyabe et al., 2001). All of the genes encoding these transporters are regulated at the transcriptional level and are induced in zinc-limited cells. This zinc-responsive gene regulation is mediated by the Zap1 transcriptional activator (Zhao and Eide, 1997). Zap1 plays a central role in zinc homeostasis by controlling the expression of these genes and ∼40 others in the yeast genome (Lyons et al., 2000). Zap1 is an 880 amino acid protein with seven C2H2 motifs. At its C-terminus is a DNA binding domain consisting of five C2H2 zinc fingers (designated ZF3–ZF7) (Figure 1) (Bird et al., 2000a; Evans-Galea et al., 2003). This domain binds specifically to a DNA element, the 11 bp zinc-responsive element or ZRE, found in one or more copies in the promoters of Zap1's target genes (Zhao et al., 1998; Lyons et al., 2000). All five of the zinc fingers in the DNA binding domain are required for ZRE interaction. Zap1 also contains two activation domains that are rich in acidic residues (Bird et al., 2000b). One activation domain, called AD1, was mapped between amino acids 330 and 552. The second activation domain, AD2, was mapped between 552 and 705. Two additional zinc fingers, ZF1 and ZF2, are found within this latter region. ZF1 and ZF2 are not required for DNA binding, highlighting a possible role as zinc sensors. Figure 1.A depiction of the Zap1 protein. The positions of the seven Zap1 zinc fingers are shown with filled boxes and are numbered. The DNA binding domain (DBD) requires fingers 3–7. Zap1's two activation domains (AD1 and AD2) are shown with hatched boxes; the location of AD2 reflects the detailed mapping data from Figure 2. The lower panel shows the sequence of Zap1 fingers 1 and 2 (residues 579–641). The positions of the β-strands and α-helices are indicated. Residues conserved in other zinc fingers are shown below the Zap1 sequence; ψ, hydrophobic. The C and G 'finger core' residues (C590 and G627) are boxed, the α-helical residues that also contribute to the hydrophobic core are underlined, and the residues proposed to make interfinger contacts are circled. Download figure Download PowerPoint Recent results have indicated that Zap1 is regulated by zinc via four different mechanisms (Zhao and Eide, 1997; A.Bird, E.Blankman, D.R.Winge and D.J.Eide, in preparation). First, Zap1 controls its own expression through transcriptional autoregulation. Secondly, zinc controls Zap1 DNA binding activity. Overexpressing Zap1 overrides DNA binding control and results in constitutive ZRE occupancy. Under these conditions, we also found that zinc independently controls the activities of AD1 and AD2. In this report, we provide a molecular model for the zinc regulation of AD2. Our results demonstrate that ZF1 and ZF2 are critical for zinc regulation of AD2 and suggest a role for these fingers in zinc sensing and consequent regulation of Zap1 activity. Results A previous study mapped AD2 to the region of Zap1 between residues 552 and 705 (Bird et al., 2000b). Located within the 552–705 region are two C2H2-type zinc finger domains designated ZF1 and ZF2 (Figure 1) that are not involved in DNA binding (Bird et al., 2000a). These two domains have most of the conserved amino acids found in other zinc fingers. The consensus sequence for these domains is Ψ/Y-X-C-X2,4-C-X3-F-X5-Ψ-X2-H-X3-5-H, where Ψ denotes a hydrophobic amino acid (Berg and Godwin, 1997). ZF1 and ZF2 match this consensus, with the exception of C (C590) and G (G627) residues located in the position most commonly occupied by F at the end of the β2 strand of fingers 1 and 2, respectively. In most zinc fingers, this residue contributes to a hydrophobic core formed by the fold between the β2 strand and the α-helix. The other residue contributing to this hydrophobic core is the conserved hydrophobic residue in the α-helix; this position is conserved in ZF1 and ZF2 (L596 and I633, respectively). The association of ZF1 and ZF2 with the zinc-responsive AD2 activation domain suggested a role for these fingers in zinc sensing. Also consistent with this hypothesis, we found that AD2 function mapped to ZF2. This detailed mapping was performed using fusions of various portions of the Zap1 552–705 region to the Gal4 DNA binding domain (GBD). Expression of a GAL1-lacZ reporter in a gal4Δ mutant strain was then used to assess activation domain function of these fusions (Figure 2). The high level of activation domain function seen with the 552–705 fragment mapped completely to the subregion of amino acids 611–641, i.e. almost the precise endpoints of ZF2 (Figure 1). No activation domain function was detected in ZF1 or elsewhere in the 552–705 region. Figure 2.Mapping AD2 within the 552–705 region. The indicated regions of Zap1 were fused to the GBD and expressed in gal4Δ cells (ABY29) co-transformed with the GAL1-lacZ reporter. Cells were grown to exponential phase in low zinc conditions (LZM + 3 μM ZnCl2) prior to β-galactosidase activity assays. A representative experiment is shown and each value is the mean of three replicates. Error bars represent 1 SD. Download figure Download PowerPoint To test whether ZF1 and ZF2 bind zinc, we determined the zinc stoichiometry, affinity and stability of zinc binding in a Zap1 fragment (amino acids 575–643) containing these fingers. For comparison, we also examined Zn2+ binding to a fragment (residues 700–766) containing ZF3 and ZF4 from the Zap1 DNA binding domain. ZF3 and ZF4, both required for binding of Zap1 to DNA in zinc-limited cells, are likely to be representative of high affinity zinc sites in other zinc-dependent proteins (e.g. TFIIIA). Following their purification from E.coli, both ZF1/ZF2 and ZF3/ZF4 fragments were found to have zinc bound with a stoichiometry of ∼2 mol eq of Zn2+ [2.3 ± 0.6 and 2.1 ± 0.6 (n = 4) for ZF1/ZF2 and ZF3/ZF4, respectively]. These results suggested that the metal was bound by both fingers in each polypeptide fragment. To determine the relative affinity of ZF1/ZF2 verses ZF3/ZF4 peptides for Zn2+, we used a competition assay with the fluorescent indicator Fura-2 (VanZile et al., 2000) Fura-2 binds Zn2+ in a 1:1 complex with a dissociation constant of 3 nM (Atar et al., 1995). Upon Zn binding to Fura-2, an absorbance shift occurs in the maxima from ∼369 to ∼339 nm, with the difference spectrum showing maximal loss of absorbance at ∼381 nm and maximal increase of absorbance at ∼332 nm (data not shown). Figure 3 shows the results of representative titration of ZnCl2 into a solution of 15 μM Fura-2, 10 μM apo-protein, 100 mM Tris–Cl, pH 7.5, with the fits calculated by the program DYNAFIT (Kuzmic, 1996). The best fits for both ZF1/ZF2 and ZF3/ZF4 are consistent with each peptide containing two Zn-binding sites of differing affinity. The apparent KD values for ZF1/ZF2 sites are 5.3 ± 2.2 and 0.3 ± 0.1 nM. The apparent KD values for ZF3/ZF4 sites are 3.0 ± 1.7 and 0.2 ± 0.0 nM. Figure 3.Determining the affinity of Zn2+ binding by Zap1 zinc fingers. The ability of ZF1/ZF2 (top panel) or ZF3/ZF4 (bottom panel) peptides to compete with the indicator Fura-2 for Zn2+ was tracked by following the loss of absorbance at 381 nm (squares indicate decreasing apo-Fura-2 concentration) and the increase of absorbance at 332 nm (circles indicate increasing Zn-Fura-2 concentration). The solution contained 15 μM Fura-2, 10 μM apo-protein, 100 μM Tris–Cl, pH 7.5. The Zn2+ titrations were performed by adding 4 μl aliquots of ZnCl2 by Hamilton syringe through an oxygen-free sealed cuvette septum. After mixing, the absorbance spectrum was scanned from 240 to 560 nm before the next titration. The final absorbance values and Zn2+ concentrations were corrected for dilution. The data was fit by the program DYNAFIT with all parameters assigned except the dissociation constants of the zinc finger pairs. A representative of three independent experiments is shown in each panel. Download figure Download PowerPoint Because the finger pairs exhibited similar affinities for Zn2+, we then assessed whether the finger pairs differed in relative stabilities of zinc binding. First, these Zn2+–zinc finger complexes were extensively dialyzed against buffer or buffer plus a zinc chelator, 1,10-phenanthroline [stability constants of 1012.2/M and 1017.1/M for the Zn(phen)2 and Zn(phen)3 complexes, respectively] (NIST Database 46: Critical Stability Constants; http://www.nist.gov/srd/nist46.htm) or a related compound, 1,7-phenanthroline, which does not bind zinc. The amount of zinc retained by these peptides after dialysis was then determined (Figure 4). After 1 day of dialysis, zinc was largely retained by both ZF1/ZF2 and ZF3/ZF4 peptides. After dialysis for 2 days, little if any zinc was removed from the ZF3/ZF4 peptide during dialysis in buffer or buffer plus 1,7-phenanthroline. The chelator removed only ∼40% of the zinc from the ZF3/ZF4 fragment under the same conditions. In contrast, dialysis of ZF1/ZF2 in buffer alone or 1,7-phenanthroline removed 80% of the bound zinc. Dialysis of ZF1/ZF2 in 1,10-phenanthroline removed almost all of the zinc. The resulting Zn-depleted ZF1/ZF2 peptide was poorly soluble. Similar results were obtained by dialysis of the ZF1/ZF2 and ZF3/ZF4 fragments against another zinc chelator, 4-(2)-(pyridylazo)resorcinol (PAR, stability constant = 1017.1/M). Dissociation of Zn2+ from ZF1/ZF2 in the presence of PAR had a t1/2 of 1.4 days while zinc loss from ZF3/ZF4 was much slower (t1/2 >14 days) (data not shown). These data indicate that ZF1 and ZF2 do bind zinc but less stably than ZF3 and ZF4. Figure 4.Lability of zinc binding by ZF1/ZF2 and ZF3/ZF4 peptides. Peptides containing the indicated fingers and with 2 mol eq Zn2+ bound initially were extensively dialyzed against buffer alone (B) or buffer containing the indicated compound (1,7P, 1, 7-phenanthroline; 1,10P, 1,10-phenanthroline) (10 μM). After dialysis for 1 or 2 days, the zinc and protein content was determined. One hundred percent is defined as zinc content of the sample prior to dialysis. The averages of two sample experiments are shown and the error bars represent ±1 SD. Download figure Download PowerPoint To further explore the stability of metal binding by Zap1 zinc finger domains, Co2+ was titrated into apo-peptides of ZF1/ZF2 and ZF3/ZF4. The energies of the d–d transitions in the visible range were consistent with the predicted C2H2 coordination (Figure 5A). Displacement of this bound Co2+ by Zn2+ can then be used to assess the kinetic stability of metal binding (Buchsbaum and Berg, 2000). Therefore, these Co2+–peptide complexes were incubated with 2 mol eq Zn2+ and the kinetics of the Zn2+-displacement of the Co2+ d–d transitions were monitored at 644 nm. As can be seen in Figure 5B (curve 1), the Co2+ ions in ZF1/ZF2 were rapidly displaced by added Zn2+ with significant displacement occurring during the mixing time prior to the first measurement (0–10 s). The rate of Co2+ displacement from ZF1/ZF2 fit well to a single exponential with a t1/2 of 5.9 s (Figure 5B, inset). In contrast, the Zn2+-induced displacement of Co2+ in the ZF3/ZF4 peptide was much slower (Figure 5B, curve 3). The t1/2 determined from the fit to a single exponential was ∼441 s under the conditions where [Co2+]tot = [Zn2+]tot. Even when the displacement of Co2+ by Zn2+ was driven by a 10-fold higher concentration of Zn2+ than Co2+ (curve 2), the t1/2 for ZF3/ZF4 sample was ∼253 s, i.e. significantly longer than the equilibration t1/2 of ZF1/ZF2 (Figure 5B, inset). Attempts to exchange Co2+ into Zn2+–ZF1/ZF2 or Zn2+–ZF3/ZF4 complexes were not successful, confirming that Zn2+ binds more avidly to the Zap1 fingers than Co2+. Figure 5.Co2+ and Zn2+ titration of Zap1 ZF1/ZF2 and ZF3/ZF4 peptides. (A) Spectra of Co2+-ZF1/ZF2 and Co2+-ZF3/ZF4 were measured with samples containing 100 μM Co2+ and 50 μM of the indicated peptide. Addition of Co2+ to either metal-free ZF1/ZF2 or ZF3/ZF4 peptides resulted in the formation of a peak with a maximum at 644 nm and a shoulder at 580 nm. Addition of higher concentrations of Co2+ did not change these spectra (data not shown). (B) After addition of Zn2+ to a solution containing the indicated Co2+–protein complex, the loss of absorbance at 644 nm was monitored over time. The final concentrations were 100 μM Co2+, 100 μM Zn2+, 50 μM ZF1/ZF2 peptide (curve 1), 100 μM Co2+, 100 μM Zn2+, 50 μM ZF3/ZF4 peptide (curve 3), or 100 μM Co2+, 1000 μM Zn2+, 50 μM ZF3/ZF4 peptide (curve 2). The data were fit to single exponential curves (insets), giving exchange t1/2 values of ∼5.9 s for ZF1/ZF2 (curve 1), ∼441 s for ZF3/ZF4 (curve 3) or ∼253 s for ZF3/ZF4, where the exchange is driven by extremely high [Zn2+] (curve 2). Download figure Download PowerPoint Their greater lability of Zn2+ binding is consistent with ZF1 and ZF2 acting as zinc sensors in regulating AD2 function. If this hypothesis is correct, mutations predicted to block zinc binding by ZF1 and/or ZF2 would also impair zinc-responsive gene regulation. GBD–Zap1552−705 fusions provided a useful assay to determine the effects of ZF1 and ZF2 mutations on zinc regulation of AD2 function. Previous studies had suggested that repression of AD2 by zinc required the presence of the Zap1 DNA binding domain (Bird et al., 2000b). Upon re-examination, we found that AD2 is regulated by zinc independently of other domains of Zap1 (see Discussion). As shown in Figure 6A, the wild-type GBD–Zap1552−705 fusion was active in zinc-limited cells and completely repressed in low zinc medium (LZM) medium supplemented with 30 μM ZnCl2. To test the role of ZF1 and ZF2 in this regulation, we first introduced mutations in which the two histidyl ligands were substituted with glutamines (i.e. C2H2→C2Q2). Such mutations, which disrupt zinc binding in other zinc fingers (Bird et al., 2000a), were generated in either ZF1, ZF2 or both. Cells expressing these C2Q2 mutant GBD–Zap1552−705 fusion proteins displayed strong activation domain function in low zinc (Figure 6A). However, in contrast to wild type, the activity of the mutant proteins was not repressed by zinc. These data suggest that zinc binding by both ZF1 and ZF2 is required for repression of AD2 function. The 2-fold increase in activity observed with zinc repletion of strains expressing the mutant fusions is similar to that seen with promoters not regulated by zinc (e.g. HIS4, CYC1) (Zhao and Eide, 1996a) and probably reflects a general decrease in expression in zinc-deficient cells that is alleviated as zinc levels rise to repletion. Figure 6.Strain ABY29 (gal4Δ) was transformed with the GAL1-lacZ reporter and plasmids expressing the indicated ZAP1 mutations in the GBD–Zap1552–705 fusion protein or the vector-only control. These cells were grown to exponential phase in LZM medium plus the indicated concentration of ZnCl2. Representative experiments are shown and each value is the mean of three replicates. Error bars represent ±1 SD. Download figure Download PowerPoint We also sought to disrupt zinc binding in ZF1 and ZF2 by generating mutants in which the C590 and G627 finger core residues (Figure 1) were substituted with other amino acids. Studies of other zinc finger peptides have shown that mutations altering this residue greatly increase the flexibility of the domain and lower the stability of Zn2+ binding (Berg and Godwin, 1997). ZF1 and ZF2 mutations were constructed in which large, charged residues (i.e. C,G→E,E; C,G→R,R) were substituted into the finger core position. These changes are likely to disrupt zinc binding by destabilizing the protein fold. When assayed for zinc responsiveness in vivo, these mutations also caused constitutive AD2 function (Figure 6B). Because the lability of zinc binding by wild type ZF1 and ZF2 could be due to the non-canonical C590 and G627 finger core residues, we also substituted these residues with phenylalanines, the residue most commonly found in this position in other zinc finger proteins. If the function of the finger core C,G residues was to reduce the zinc binding stability of ZF1 and ZF2, we predicted that the C,G→F,F substitutions would be repressed by even lower concentrations of Zn2+ than the wild-type fragment. In vivo, the ZF1 and ZF2 C,G→F,F mutant was regulated similar to wild type (Figure 6B). These results indicate that the non-canonical C and G finger core residues of ZF1 and ZF2 do not solely determine a regulatory set-point of these fingers. Immunoblotting indicated that the wild-type fusion and the C,G→F,F mutant proteins accumulated to similarly high levels (data not shown). Surprisingly, the mutants predicted to be defective for Zn2+ binding were destabilized in vivo such that steady-state protein levels were lower. Therefore, the failure of these mutant proteins to be regulated by zinc is not due to their overexpression relative to the wild-type fusion. The results shown in Figure 6A indicate that while AD2 function mapped to ZF2, both ZF1 and ZF2 are required to repress AD2. One explanation for this requirement is that an intramolecular interaction occurs between these two fingers to form a conformation that represses AD2 function. That such a finger–finger interaction occurs was suggested by the amino acid sequence of these domains. Zap1 fingers 1 and 2 resemble the first two of the five zinc fingers in the Gli protein (Pavletich and Pabo, 1993). These two Gli fingers make intramolecular protein–protein contacts with each other. The packing interface between these fingers consists of two W residues in the β-hairpin loops between the cysteinyl ligands and hydrophobic packing between the two α-helices. The β-hairpin loops of Zap1 ZF1 and ZF2 contain similarly positioned W residues (W583 and W620) and the nonpolar residues contributing to interhelical hydrophobic packing in Gli are also conserved in Zap1 ZF1 and ZF2 (L600, V605, V634, I637) (Figure 1). To test the hypothesis that ZF1 and ZF2 interact to mask AD2, we constructed mutant alleles of ZF1 predicted to disrupt this proposed interaction. First, W583 was mutated to alanine. The adjacent K582 was also mutated to A in this allele (K582A, W583A) because it could potentially form an interaction-stabilizing salt bridge with E621 of ZF2. In a second mutant, the two hydrophobic residues in the α-helix of ZF1, L600 and V605, were mutated to aspartates (L600D, V605D). The effects of these mutations on AD2 regulation were determined using GBD–Zap1552−705 fusions. As predicted if zinc-responsive repression of AD2 required an interaction between ZF1 and ZF2, these mutations totally disrupted zinc regulation of AD2 activity (Figure 6C). Similar zinc non-responsiveness was observed when the W and adjacent E residues in ZF2 predicted to participate in the interaction were mutated (W620A, E621K) (data not shown). The analysis of AD2 regulation presented thus far has considered the behavior of AD2 in the non-native context of GBD–Zap1 fusions. If ZF1 and ZF2 are indeed involved in zinc regulation of Zap1, mutations affecting these domains should have some effect on the zinc-responsiveness of the full-length Zap1 protein. We introduced the ZF1/ZF2 C2Q2 mutations into full-length Zap1 expressed from the GAL1 promoter. The GAL1 promoter allows assessment of Zap1 function at low levels of expression (i.e. in glucose-grown cells) where the independent control of DNA binding by zinc occurs, and at high Zap1 levels (i.e. in galactose-grown cells) where ZRE occupancy is constitutive (A.Bird, E.Blankman, M.Evans-Galea, D.R.Winge and D.J.Eide, in preparation). These experiments were performed in a zap1Δ strain to allow analysis of the activity of the plasmid-encoded allele on a ZRE-lacZ reporter. Figure 7A shows Zap1 activity when DNA binding control is zinc-responsive. In the context of full-length Zap1, the ZF1/ZF2 C2Q2 mutant showed a reproducible defect in zinc repression at media zinc concentrations from 30 to 300 μM. The scale in Figure 7A tends to obscure the magnitude of this effect. As shown in Figure 7A (inset), expression at these concentrations (e.g. LZM + 300 μM ZnCl2) can be almost 3-fold higher in the mutant than the wild type. In the absence of DNA binding control, i.e. when ZRE occupancy is rendered constitutive by Zap1 overexpression, an even more striking result was obtained (Figure 7B). Little or no repression of the mutant Zap1 activity by zinc was observed up to 300 μM zinc, while the wild type showed repression with as little as 30 μM. These results demonstrate that ZF1 and ZF2 are important for the zinc responsiveness of Zap1 when the protein is bound to DNA. The residual regulation observed for the constitutively bound mutant protein may be largely due to regulation of AD1, independent of AD2 (Bird et al., 2000b). Figure 7.Effects of ZF1/ZF2 mutations on the zinc responsiveness of full-length Zap1. ZHY6 (zap1Δ) cells transformed with the ZRE-lacZ (pDg2) reporter and pYef2 (vector), full-length wild-type Zap1 (pMyc-Zap1), or full-length Zap1 with the ZF1/ZF2 C2Q2 mutations (pZF1/21–880) were grown in LZM medium plus the indicated concentration of ZnCl2. (A) Low-level expression of Zap1 from the GAL1 promoter in glucose-grown cells. DNA binding control occurs normally under these conditions. The inset shows the data for these strains grown in LZM + 300 μM ZnCl2. The asterisks indicate a significant difference from wild type (P < 0.05) as estimated by ANOVA. (B) High-level expression of Zap1 from the GAL1 promoter in galactose-grown cells. ZRE occupancy is constitutive under these conditions. A representative experiment of three separate experiments is shown and each value is the mean of three replicates. Error bars represent ±1 SD. Download figure Download PowerPoint Finally, we examined the effects of ZF1/ZF2 C2Q2 mutations on zinc-responsiveness when Zap1 was expressed from its own promoter. A strain was engineered in which the ZF1/ZF2 C2Q2 mutations were introduced into the chromosomal ZAP1 gene. In this context, Zap1 expression is subject to transcriptional autoregulation, DNA binding control, and control of AD1 and AD2. Zinc-responsive gene expression was assayed by measuring mRNA levels generated by the chromosomal ZRT1 gene by S1 nuclease protection assay. In wild-type cells, ZRT1 expression was greatly repressed by zinc at concentrations of 30 μM and higher (Figure 8A). ZRT1 mRNA levels were quantified along with CMD1 (calmodulin) as a loading control. The ratio of ZRT1:CMD1 mRNA is plotted in Figure 8B. In contrast, zinc regulation of the chromosomal ZAP1ZF1&2 C2Q2 mutant was greatly impaired. These results demonstrate the importance of ZF1 and ZF2 in regulating Zap1 activity on a native target promoter. Figure 8.Effects of ZF1/ZF2 mutations in the chromosomal ZAP1 gene on the regulation of gene expression. (A) Total RNA was extracted from exponential-phase cultures of the zap1 mutant strain ZHY6 (Δ) grown in LZM media supplemented with 3000 μM Zn2+ (lane 1) and from the wild-type strain, DY1457 and a chromosomal ZAP1ZF1&2 C2Q2 mutant, grown in LZM media supplemented with 3, 10, 30, 100, 300, 1000 and 3000 μM Zn2+ (lanes 2–8, respectively). The levels of ZRT1 mRNA were compared to the loading control CMD1 mRNA using S1 nuclease protection assays. Arrows indicate ZRT1 and CMD1 S1 nuclease protection products. (B) The band intensities in (A) were quantified and are plotted as the ratio of ZRT1:CMD1 mRNA levels at each zinc concentration. A representative of
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