Induction of the ZRC1 Metal Tolerance Gene in Zinc-limited Yeast Confers Resistance to Zinc Shock
2003; Elsevier BV; Volume: 278; Issue: 17 Linguagem: Inglês
10.1074/jbc.m300568200
ISSN1083-351X
AutoresColin W. MacDiarmid, Mark A. Milanick, David Eide,
Tópico(s)Plant Micronutrient Interactions and Effects
ResumoZinc is an essential nutrient but toxic to cells with overaccumulation. For this reason, intracellular zinc levels are tightly controlled. In the yeast Saccharomyces cerevisiae, the Zrc1 and Cot1 proteins have been implicated in the storage and detoxification of excess zinc in the vacuole. Surprisingly, transcription of ZRC1 is induced in zinc-limited cells by the zinc-responsive transcription factor Zap1. We show here that this increase in ZRC1 expression is a novel mechanism of zinc homeostasis and stress tolerance. Zinc-limited cells also express high levels of the plasma membrane zinc uptake transporters. As a consequence, when zinc-limited cells are resupplied with small amounts of zinc, large quantities quickly accumulate in the cell, a condition we refer to as "zinc shock." We show here that ZRC1 and its induction in zinc-limited cells are required for resistance to this zinc shock. Experiments using the zinc-responsive fluorophore FuraZin-1 as an indicator of vacuolar zinc levels indicated that Zrc1 is required for the rapid transport of zinc into the vacuole during zinc shock. We also present evidence that cytosolic zinc rises to higher levels in cells unable to sequester this excess zinc. Thus, the increase in ZRC1 expression occurs prior to the zinc shock stress for which this induction is important. We propose that this "proactive" strategy of homeostatic regulation, such as we document here for ZRC1, may represent a common but largely unrecognized phenomenon. Zinc is an essential nutrient but toxic to cells with overaccumulation. For this reason, intracellular zinc levels are tightly controlled. In the yeast Saccharomyces cerevisiae, the Zrc1 and Cot1 proteins have been implicated in the storage and detoxification of excess zinc in the vacuole. Surprisingly, transcription of ZRC1 is induced in zinc-limited cells by the zinc-responsive transcription factor Zap1. We show here that this increase in ZRC1 expression is a novel mechanism of zinc homeostasis and stress tolerance. Zinc-limited cells also express high levels of the plasma membrane zinc uptake transporters. As a consequence, when zinc-limited cells are resupplied with small amounts of zinc, large quantities quickly accumulate in the cell, a condition we refer to as "zinc shock." We show here that ZRC1 and its induction in zinc-limited cells are required for resistance to this zinc shock. Experiments using the zinc-responsive fluorophore FuraZin-1 as an indicator of vacuolar zinc levels indicated that Zrc1 is required for the rapid transport of zinc into the vacuole during zinc shock. We also present evidence that cytosolic zinc rises to higher levels in cells unable to sequester this excess zinc. Thus, the increase in ZRC1 expression occurs prior to the zinc shock stress for which this induction is important. We propose that this "proactive" strategy of homeostatic regulation, such as we document here for ZRC1, may represent a common but largely unrecognized phenomenon. zinc response element 4-morpholineethanesulfonic acid synthetic-defined medium or standard deviation chelex-treated synthetic-defined medium low zinc medium wild type All organisms face constantly changing nutrient availability and environmental stresses. As a consequence, organisms have regulatory mechanisms that maintain nutrient homeostasis and deal with damage caused by stress. For example, deficiency of an essential nutrient often induces expression of the genes involved in acquiring that nutrient. Stresses such as heat shock or reactive oxygen species increase expression of genes whose products reverse the resultant cellular damage. As a rule, these regulatory systems are reactionary in nature, responding to the stress to alter levels of gene expression and/or protein activity. An alternative strategy would be to increase expression of the required genes prior to, rather than in response to the specific stress. We designate this latter strategy as "proactive" regulation to distinguish it from the commonly recognized "reactive" responses. In this report, we describe an apparent proactive mechanism of zinc homeostasis in the yeastSaccharomyces cerevisiae, the up-regulation of a metal tolerance gene in zinc-deficient cells as protection against "zinc shock." Zinc is an essential nutrient required for many processes. Its chemical properties make this metal a useful catalytic and/or structural cofactor in many proteins. Despite its importance however, excess zinc is toxic. Zinc toxicity may involve competition with other metal ions for the active sites of enzymes or intracellular transport proteins. For this reason, organisms have evolved with mechanisms of zinc homeostasis that tightly control the intracellular level of zinc as extracellular concentrations change. An indication of the exquisite precision of this control was recently obtained in studies ofEscherichia coli, where regulatory systems controlling zinc uptake and efflux apparently strive to maintain the free intracellular zinc concentration at less than one atom per cell (1Outten C.E. O'Halloran T.V. Science. 2001; 292: 2488-2492Crossref PubMed Scopus (1173) Google Scholar). Several reports suggest that eukaryotic cells also maintain very low levels of cytoplasmic labile zinc under steady-state conditions (2Cheng C. Reynolds I.J. J. Neurochem. 1998; 71: 2401-2410Crossref PubMed Scopus (71) Google Scholar, 3Atar D. Backx P.H. Appel M.M. Gao W.D. Marban E. J. Biol. Chem. 1995; 270: 2473-2477Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 4Ragozzino D. Giovannelli A. Degasperi V. Eusebi F. Grassi F. J. Physiol. 2000; 529: 83-91Crossref PubMed Scopus (19) Google Scholar). Studies of yeast have revealed several components of zinc homeostasis in this organism (5Gaither L.A. Eide D.J. Biometals. 2001; 14: 251-270Crossref PubMed Scopus (433) Google Scholar). Among these, the ZRC1 gene encodes a potential transporter protein of the cation diffusion facilitator (CDF) family (6Paulsen I.T. Saier M.H. J. Membr. Biol. 1997; 156: 99-103Crossref PubMed Scopus (297) Google Scholar). This family also includes bacterial, plant, and mammalian proteins involved in zinc efflux and compartmentalization.ZRC1 is known to contribute to zinc tolerance (7Kamizono A. Nishizawa M. Teranishi Y. Murata K. Kimura A. Mol. Gen. Genet. 1989; 219: 161-167Crossref PubMed Scopus (164) Google Scholar, 8Conklin D.S. Culbertson M.R. Kung C. Mol. Gen. Genet. 1994; 244: 303-311Crossref PubMed Scopus (58) Google Scholar), and the Zrc1 protein was localized to the yeast vacuole membrane (9Miyabe S. Izawa S. Inoue Y. Biochem. Biophys. Res. Commun. 2001; 282: 79-83Crossref PubMed Scopus (57) Google Scholar, 10Li L. Kaplan J. J. Biol. Chem. 1998; 273: 22181-22187Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 11MacDiarmid C.W. Milanick M.A. Eide D.J. J. Biol. Chem. 2002; 277: 39187-39194Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Our recent in vitro studies provided evidence that Zrc1 directly mediates vacuolar zinc transport, most likely via a zinc/H+ antiport mechanism (11MacDiarmid C.W. Milanick M.A. Eide D.J. J. Biol. Chem. 2002; 277: 39187-39194Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). The COT1 gene encodes a related protein that may act analogously to Zrc1 in cobalt detoxification (8Conklin D.S. Culbertson M.R. Kung C. Mol. Gen. Genet. 1994; 244: 303-311Crossref PubMed Scopus (58) Google Scholar, 12Conklin D.S. McMaster J.A. Culbertson M.R. Kung C. Mol. Cell. Biol. 1992; 12: 3678-3688Crossref PubMed Scopus (181) Google Scholar). Cot1 also contributes to zinc detoxification (13Bloss T. Clemens S. Nies D.H. Planta. 2002; 214: 783-791Crossref PubMed Scopus (87) Google Scholar) and both Zrc1 and Cot1 appear to be required for sequestration of zinc in an intracellular storage compartment for later use under zinc-limiting conditions (14MacDiarmid C.W. Gaither L.A. Eide D. EMBO J. 2000; 19: 2845-2855Crossref PubMed Scopus (309) Google Scholar). During the transition from zinc-replete to zinc-limiting conditions, many genes are induced to maintain adequate supplies of zinc for cell growth. Three such genes, ZRT1, ZRT2, andFET4 encode plasma membrane transporters responsible for zinc uptake under deficient conditions (15Zhao H. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2454-2458Crossref PubMed Scopus (453) Google Scholar, 16Zhao H. Eide D. J. Biol. Chem. 1996; 271: 23203-23210Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 17Waters B.M. Eide D.J. J. Biol. Chem. 2002; 277: 33749-33757Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). All three genes are targets of the Zap1 transcriptional activator (18Zhao H. Eide D.J. Mol. Cell. Biol. 1997; 17: 5044-5052Crossref PubMed Scopus (223) Google Scholar), a central player in zinc homeostasis. Zap1 is active in zinc-limited cells and is repressed by high cytoplasmic/nuclear zinc (19Bird A.J. Zhao H. Luo H. Jensen L.T. Srinivasan C. Evans-Galea M. Winge D.R. Eide D.J. EMBO J. 2000; 19: 3704-3713Crossref PubMed Scopus (71) Google Scholar). When active, Zap1p binds to an 11-bp sequence, the zinc response element (ZRE),1 in the promoters of its target genes (20Zhao H. Butler E. Rodgers J. Spizzo T. Duesterhoeft S. Eide D. J. Biol. Chem. 1998; 273: 28713-28720Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 21Lyons T.J. Gasch A.P. Gaither L.A. Botstein D. Brown P.O. Eide D.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7957-7962Crossref PubMed Scopus (252) Google Scholar). DNA microarray analysis has suggested that at least 46 genes in yeast are direct targets of Zap1 regulation (21Lyons T.J. Gasch A.P. Gaither L.A. Botstein D. Brown P.O. Eide D.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7957-7962Crossref PubMed Scopus (252) Google Scholar). Surprisingly, this group and others (22Miyabe S. Izawa S. Inoue Y. Biochem. Biophys. Res. Commun. 2000; 276: 879-884Crossref PubMed Scopus (34) Google Scholar) noted ZRC1 among these Zap1 target genes. These data raise an intriguing question. Why would a transporter involved in zinc storage and detoxification be up-regulated under zinc-limiting conditions? One potential explanation was suggested by our previous studies of zinc uptake by zinc-deficient cells. Due to the induction of ZRT1, ZRT2, and FET4, the maximum capacity for zinc uptake by zinc-limited cells is ∼100-fold greater than in zinc-replete cells (15Zhao H. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2454-2458Crossref PubMed Scopus (453) Google Scholar, 16Zhao H. Eide D. J. Biol. Chem. 1996; 271: 23203-23210Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). Therefore, when even a low concentration of zinc is added back to zinc-limited cells, the metal is rapidly overaccumulated, a condition we refer to as zinc shock. In this report we show that the increased expression of ZRC1 in zinc-limited cells is essential for the cell's ability to tolerate the stress of zinc shock. Four different media were used for yeast cultures. YPD and synthetic-defined (S.D.) medium with 2% glucose (23Sherman F. Fink G.R. Hicks J.B. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1986Google Scholar) are zinc-replete and contain no strong chelators. Low zinc medium (LZM) and chelex-treated synthetic-defined (CSD) medium were prepared as previously described (14MacDiarmid C.W. Gaither L.A. Eide D. EMBO J. 2000; 19: 2845-2855Crossref PubMed Scopus (309) Google Scholar, 21Lyons T.J. Gasch A.P. Gaither L.A. Botstein D. Brown P.O. Eide D.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7957-7962Crossref PubMed Scopus (252) Google Scholar). LZM is zinc limiting because of the inclusion of 1 mm EDTA and 20 mmcitrate as divalent cation chelators. CSD is zinc limiting because zinc is removed from the medium with the chelex-100 ion exchange resin. Zinc supplemented into CSD is much more bioavailable than in LZM due to the absence of strong chelators in CSD. Cell density determinations and β-galactosidase assays were performed as previously described (14MacDiarmid C.W. Gaither L.A. Eide D. EMBO J. 2000; 19: 2845-2855Crossref PubMed Scopus (309) Google Scholar).S. cerevisiae strains CM100, 102, 103, 104, and 142 were also previously described (14MacDiarmid C.W. Gaither L.A. Eide D. EMBO J. 2000; 19: 2845-2855Crossref PubMed Scopus (309) Google Scholar). All newly constructed strains are isogenic to CM100. CM141 (MATα zrt1::LEU2 zrc1::HIS3) was derived from a cross of ZHY1 (15Zhao H. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2454-2458Crossref PubMed Scopus (453) Google Scholar) and CM102. The CM146 (MATa zrc1mZRE) strain was constructed by integrating the insert of YCpzrc1mZRE at the chromosomal ZRC1 locus. Transformants were selected by complementation of the zinc-sensitive phenotype of a zrc1 cot1 double mutant strain (CM104), and the presence of the mutation was verified using PCR. The cot1 mutation was then removed by backcrossing to a wild-type strain. To construct the YEpzrc1mZRE plasmid, overlap PCR was used to generate transversion mutations in the ZRE sequence of the ZRC1promoter (24Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6851) Google Scholar). The resulting fragment was inserted intoBlpI-, BstXI-digested YCpZRC1 (11MacDiarmid C.W. Milanick M.A. Eide D.J. J. Biol. Chem. 2002; 277: 39187-39194Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar) by gap repair. Construction of YEpZRC1-lacZ was previously described (21Lyons T.J. Gasch A.P. Gaither L.A. Botstein D. Brown P.O. Eide D.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7957-7962Crossref PubMed Scopus (252) Google Scholar). YEpzrc1mZRE-lacZ was constructed by amplifying theZRC1 promoter from YEpzrc1mZRE and inserting the fragment into YEp353 by gap repair. All plasmid constructs were verified by DNA sequencing. Total protein was extracted from yeast, and immunoblot analysis was performed as previously described (25Gitan R.S. Eide D.J. Biochem. J. 2000; 346: 329-336Crossref PubMed Scopus (147) Google Scholar). Anti-Vma1 antibody was obtained from Molecular Probes. Zinc uptake was assayed as described (15Zhao H. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2454-2458Crossref PubMed Scopus (453) Google Scholar). Standard zinc uptake assays were performed in LZM lacking EDTA (LZM-EDTA) to increase the bioavailability of the zinc tracer. To determine cell-associated zinc after growth in65Zn2+-containing medium, 1-ml aliquots of cells were collected on glass fiber filters (Whatman) and washed twice with 5 ml of wash buffer (20 mm sodium citrate, pH 4, 1 mm EDTA). Radioactivity retained on the filters was quantified with a gamma counter. 65Zn2+ uptake by cells loaded with FuraZin-1 (see below) was assayed in the same buffer used for assays of fluorophore fluorescence to allow direct comparison of total cell and vacuolar zinc accumulation under these conditions. Yeast cultures (250 ml) were grown to log phase in LZM + 2 μmzinc. Cells were harvested, washed twice with phosphate-buffered saline and resuspended in phosphate-buffered saline at a final density of 3 × 108 cells/ml. 50 μg of FuraZin-1 acetoxymethyl (AM) ester was dissolved in 16.6 μl of a 20% Pluronic solution (both obtained from Molecular Probes) and the solution diluted 4-fold with Me2SO to give a stock solution of 1.25 mm fluorophore and 5% Pluronic. Fluorophore was added to a 1-ml aliquot of the cell suspensions to give a final concentration of 25 μm. Another 1-ml aliquot of each strain was treated in parallel but without exposure to fluorophore. The cell suspensions were incubated at 30 °C in the dark for 1 h with agitation. The cells were recovered by centrifugation, washed three times with 5 ml of chilled zinc uptake buffer (10 mm MES-Tris, pH 6.5, 4 mm MgCl2, 2% glucose) and 1 mmEDTA and then incubated at 30 °C for another 30 min. This step allowed the cells to redistribute cytoplasmic fluorophore to the vacuole and complete its hydrolysis. The cells were then chilled and washed twice with MES-Tris uptake buffer (−EDTA). Cell pellets were resuspended in 2 ml of uptake buffer and maintained on ice prior to zinc uptake assays. Fluorimetric assays of fluorophore speciation were performed in a Hitachi F3010 spectrofluorimeter. To start the assay, an aliquot of loaded cells (100 μl) was added to 4 ml of MES-Tris uptake buffer and 100 μm zinc. The temperature of the cuvette was maintained at 30 °C using a recirculating water bath. With the instrument set at maximum scan speed, the excitation wavelength was varied from 250 to 450 nm, and the intensity of emission at 500 nm was recorded. Spectra were recorded at the start of the assay and at 2-min intervals for up to 6 min. To correct for the effects of fluorophore leakage during the experiment, immediately before each measurement a 1-ml aliquot of the assay was removed and filtered through a 0.45-μm syringe filter. After completion of the experiment, unloaded cells were added to samples of the filtered buffer to give the same cell density as present in the initial suspensions. Spectra of these samples were immediately recorded and the curves subtracted from the original spectra. This procedure provided a one-step correction for both cellular autofluorescence and leakage of fluorophore from the cells during the experiment. The corrected traces were scanned to obtain emission intensities at the excitation wavelengths of 325 and 380 nm. A previous study indicated that both Zrc1 and Cot1 contribute to tolerance of excess zinc (13Bloss T. Clemens S. Nies D.H. Planta. 2002; 214: 783-791Crossref PubMed Scopus (87) Google Scholar). In the yeast strain INVSC2, wild-type and cot1 cells were resistant to zinc concentrations up to ∼5 mm. Mutant zrc1 cells tolerated up to 1 mm, but the zrc1 cot1 mutant was not viable at this zinc concentration. In our strain background (W303), we obtained qualitatively similar results (Fig.1). Wild-type cells showed tolerance to up to 6 mm zinc. The zrc1 mutation caused a slight defect in zinc tolerance, reducing the inhibitory concentration by 1–3 mm. The cot1 mutation had little effect, only lowering the maximum tolerable concentration to 5 mm. In contrast to these minor effects, the zrc1 cot1 double mutant strain showed a ∼50-fold reduction in the tolerable zinc concentration (0.07 mm) relative to the single mutants. These observations demonstrated that Zrc1 and Cot1 are functionally redundant for protection against high levels of zinc under steady-state conditions. However, even with the loss of both genes, yeast was still tolerant of a relatively high concentration of zinc (70 μm). Previous studies suggested a role for Zrc1 in zinc-limited cells. Specifically, expression of the ZRC1 gene is induced in zinc-limited cells in a Zap1-dependent manner (21Lyons T.J. Gasch A.P. Gaither L.A. Botstein D. Brown P.O. Eide D.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7957-7962Crossref PubMed Scopus (252) Google Scholar, 22Miyabe S. Izawa S. Inoue Y. Biochem. Biophys. Res. Commun. 2000; 276: 879-884Crossref PubMed Scopus (34) Google Scholar). Furthermore, theZRC1 promoter contains a potential ZRE that is functional when inserted into a heterologous promoter (21Lyons T.J. Gasch A.P. Gaither L.A. Botstein D. Brown P.O. Eide D.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7957-7962Crossref PubMed Scopus (252) Google Scholar). To further examine this regulation, a mutant ZRC1 promoter-lacZ fusion was generated in which all nucleotides in the putative ZRE were altered by transversion mutations. Strains bearing wild-type or ZRE mutant reporter plasmids were cultured in LZM supplemented with a range of zinc levels and then assayed for β-galactosidase activity (LZM is zinc limiting because it contains EDTA, which chelates most zinc in the medium rendering it unavailable to cells). The wild-type promoter was strongly induced under zinc-limited conditions (Fig.2A). Added zinc reduced expression to ∼20% of the maximal level. This basal expression ofZRC1 is Zap1-independent (21Lyons T.J. Gasch A.P. Gaither L.A. Botstein D. Brown P.O. Eide D.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7957-7962Crossref PubMed Scopus (252) Google Scholar, 22Miyabe S. Izawa S. Inoue Y. Biochem. Biophys. Res. Commun. 2000; 276: 879-884Crossref PubMed Scopus (34) Google Scholar). Consistent with this conclusion, mutation of the ZRE completely eliminated zinc-responsive regulation without affecting basal expression. This result supports the contention that ZRC1 is transcriptionally regulated by Zap1 through this ZRE. Furthermore, the ZRE mutant promoter provided a useful reagent for subsequent studies (see below). Transcriptional control of ZRC1 was also reflected in altered protein levels. We examined the accumulation of protein expressed from a functional (data not shown) epitope-tagged allele ofZRC1 regulated by its own promoter and integrated into its native chromosomal location. Immunoblot analysis showed that two closely spaced bands representing forms of the Zrc1 protein accumulated to higher levels in zinc-deficient cells (LZM + 0.3–10 μm zinc) compared with zinc-replete cells (Fig.2B). No bands of this size were detected in protein from a control strain expressing untagged Zrc1 (data not shown). The level of a loading control protein, Vma1, was minimally affected by zinc status. The Zap1-dependent regulation ofZRC1 suggested that the Zrc1 protein is required during zinc deficiency. Supporting this hypothesis, it was previously reported thatzrc1 mutants grew poorly compared with wild type under zinc-limiting conditions (22Miyabe S. Izawa S. Inoue Y. Biochem. Biophys. Res. Commun. 2000; 276: 879-884Crossref PubMed Scopus (34) Google Scholar). Contrary to that previous result, however, we found no defect in zinc-limited growth (LZM + 0.3–10 μm zinc) for zrc1, cot1, orzrc1 cot1 cells (Fig.3A). The explanation for the apparent discrepancy between our results and the prior study will be presented under "Discussion." The sensitivity of the zrc1 cot1 mutant to high zinc (LZM + 30–1000 μm zinc) is consistent with the redundancy of Zrc1 and Cot1 in steady-state zinc tolerance. To determine the effect of these mutations on zinc accumulation, we measured the total zinc content of wild-type and mutant strains after growth over a range of added zinc. Again, at zinc concentrations of 10 μm or less, these mutations had no effect on cell zinc content (Fig. 3B). In medium supplemented with 30 μm zinc or more, all three mutant strains accumulated less zinc than wild type, and the zrc1 cot1 double mutant accumulated less than either single mutant. Notably, these effects of zrc1 and cot1 mutations on growth and zinc accumulation were observed only at zinc concentrations in LZM of greater than 10 μm. This concentration was previously identified as the transition point between zinc deficiency and repletion (14MacDiarmid C.W. Gaither L.A. Eide D. EMBO J. 2000; 19: 2845-2855Crossref PubMed Scopus (309) Google Scholar). This can also be seen as such here because for the wild-type strain, the maximum growth rate is observed at 10 μm zinc and higher. Thus, we found no evidence that Zrc1 or Cot1 are required for zinc-limited growth or alter zinc homeostasis in zinc-limited cells. To test for an effect of high ZRC1 activity on zinc homeostasis in zinc-deficient cells, we overexpressed the gene from a multicopy plasmid and measured the effect on total cellular zinc accumulation. We confirmed an increase in Zrc1 protein level due to overexpression in both high and low zinc media (data not shown). As shown in Fig. 4A, overexpression of ZRC1 significantly increased cellular zinc content when cells were grown under zinc-replete conditions. Importantly, it had no effect in zinc-deficient conditions (Fig.4A, inset). The effect of this increased zinc accumulation on cytoplasmic zinc availability was then determined. Methods to directly measure cytoplasmic labile zinc are not currently available, but we have previously shown that an indirect assessment of this parameter can be made using a Zap1-regulated reporter gene (14MacDiarmid C.W. Gaither L.A. Eide D. EMBO J. 2000; 19: 2845-2855Crossref PubMed Scopus (309) Google Scholar). ZRE-lacZ reporter activity was significantly increased in cells that overexpressed ZRC1, indicating reduced cytoplasmic labile zinc. This observation is consistent with a model whereby Zrc1 expression results in zinc transport from the cytoplasmic/nuclear compartment into an organelle. Again however, an effect of Zrc1 was only seen in cells that were zinc-replete. In summary, the experiments shown in Figs. 3 and 4 revealed no evidence for an effect of Zrc1 on zinc homeostasis in yeast cells during growth under steady-state zinc-deficient conditions. Thus, while it is not possible to rule out a more subtle role not revealed by these assays, these experiments did not provide an obvious explanation for why ZRC1 is up-regulated in zinc-deficient cells. In the experiments shown in Fig. 1, zinc-replete cells were used to inoculate plates. Zinc-replete cells have repressed high-affinity zinc uptake systems and do not accumulate excess zinc when inoculated into fresh medium. In contrast, when zinc-limited cells are resupplied with zinc, they rapidly accumulate large quantities because of the high activity of the plasma membrane zinc transporters (15Zhao H. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2454-2458Crossref PubMed Scopus (453) Google Scholar). We refer to this condition as zinc shock. During zinc shock, newly acquired zinc rapidly enters the cytoplasm where it can accumulate to high levels. We hypothesized that yeast detoxify this excess zinc by rapid transport into an intracellular compartment. If this is true, the high expression of the ZRC1 zinc tolerance gene in zinc-deficient cells may be required to mediate the rapid sequestration of excess cytoplasmic zinc during zinc shock. This model predicts that azrc1 mutant will be hypersensitive to zinc shock; a prediction we confirmed (Fig.5A). The medium used in this experiment, CSD, is made zinc limiting by treatment with a chelating resin. Because CSD contains no strong chelators, the availability of added zinc is many orders of magnitude higher than for equivalent concentrations of total zinc in LZM. When zinc-limited zrc1mutants (i.e. pregrown in LZM + 1 μmZnCl2) were inoculated into CSD medium supplemented with as little as 1 μm zinc, zrc1 cells failed to grow. In contrast, neither the wild-type nor the cot1 mutant showed any growth defect up to 10 μm zinc (the increase in growth yield observed for the wild-type and cot1 strains with increased zinc is due to zinc-limitation in CSD with less than 1 μm added zinc). The zrc1 cot1 mutant was even more sensitive to zinc shock than the zrc1 mutant, indicating Cot1 also contributes to zinc shock tolerance. None of these cell types were zinc sensitive when inoculated from zinc-replete cultures (i.e. pregrown in LZM + 1 mmZnCl2) (Fig. 5B). Thus, the sensitivity is a consequence of the transition from zinc-limited to zinc-replete conditions. The retarded growth of the zrc1 cot1 mutant when inoculated from zinc-replete under zinc-limiting conditions does not correspond to zinc toxicity (because growth at higher zinc concentrations is similar to wild-type), and may be due to the low vacuolar zinc stores in this mutant (14MacDiarmid C.W. Gaither L.A. Eide D. EMBO J. 2000; 19: 2845-2855Crossref PubMed Scopus (309) Google Scholar). While the Zrt1, Zrt2, and Fet4 plasma membrane uptake transporters are induced in zinc-limited cells, Zrt1 is the major pathway of zinc uptake; mutation of ZRT1 reduces zinc uptake in zinc-deficient cells by ≥80% (16Zhao H. Eide D. J. Biol. Chem. 1996; 271: 23203-23210Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). Therefore, we predicted that zinc shock results largely from the high activity of Zrt1 in deficient cells. To test this prediction, we examined the effect of azrt1 mutation on the zinc sensitivity of a zrc1mutant. As before, zinc-deficient cells were inoculated into CSD medium containing no zinc or 1 μm added zinc. Thezrt1 mutation completely suppressed the zinc sensitivity associated with the zrc1 mutation (Fig. 5C). These data indicate that the high level of zinc accumulation mediated by Zrt1 in zinc-limited cells is responsible for the zinc sensitivity of zrc1 mutants undergoing zinc shock. Poor growth of thezrt1 and zrt1 zrc1 mutants without added zinc is likely due to the impaired zinc uptake in these strains. To assess if zinc sequestration is altered in zrc1 and cot1 mutants during zinc shock, we first assayed the effects of these mutations on zinc accumulation. Accumulation of substrate on the cytoplasmic side of the plasma membrane can directly inhibit the transporters responsible for uptake, for example via trans-inhibition (26Stein W.D. Channels, carriers, and pumps: an introduction to membrane transport. Academic Press, San Diego1990: 199-200Google Scholar, 27Grenson M. Pont J.J.H.H.M.D. Molecular aspects of transport proteins. Elsevier Science Publishers, Amsterdam1992: 219-245Google Scholar). Data from zinc uptake experiments (15Zhao H. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2454-2458Crossref PubMed Scopus (453) Google Scholar) predicted that in the absence of any intracellular compartmentalization during zinc shock, cytoplasmic zinc levels would rise into the millimolar range within minutes. Under these conditions, mutant strains unable to sequester zinc might exhibit impaired zinc uptake due to trans-inhibition. To test this prediction, zinc accumulation by wild-type, zrc1,cot1, and zrc1 cot1 mutants under zinc shock conditions was compared; i.e. zinc-deficient cells were transferred to an uptake buffer that c
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