Zinc-induced Inactivation of the Yeast ZRT1 Zinc Transporter Occurs through Endocytosis and Vacuolar Degradation*

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

The ZRT1 gene encodes the transporter responsible for high affinity zinc uptake in yeast. ZRT1 is transcribed in zinc-limited cells and its transcription is repressed in zinc-replete cells. In this report, we describe a second, post-translational mechanism that regulates ZRT1 activity. In zinc-limited cells, ZRT1 is a stable, N-glycosylated plasma membrane protein. Exposure to high levels of extracellular zinc triggers a rapid loss of ZRT1 uptake activity. Our results demonstrate that this inactivation occurs through zinc-induced endocytosis of the protein and its subsequent degradation in the vacuole. Mutations that inhibit the internalization step of endocytosis also inhibited zinc-induced ZRT1 inactivation and the major vacuolar proteases were required to degrade ZRT1 in response to zinc. Furthermore, immunofluorescence microscopy showed that ZRT1 is localized to the plasma membrane in zinc-limited cells and that the protein is transferred to the vacuole via an endosome-like compartment upon exposure to zinc. ZRT1 inactivation is a relatively specific response to zinc; cadmium and cobalt ions trigger the response but less effectively than zinc. Moreover, zinc does not alter the stability of several other plasma membrane proteins. Therefore, zinc-induced ZRT1 inactivation is a specific regulatory system to shut off zinc uptake activity in cells exposed to high extracellular zinc levels thereby preventing overaccumulation of this potentially toxic metal.
The plasma membrane is an essential barrier between the extracellular milieu and the interior of the cell. The large number of receptors and transport proteins embedded in this membrane facilitate communication between the exterior and interior of the cell, selective accumulation of nutrients, and efflux of ions and other substrates. Controlling the activity of these receptors and transporters is a critical part of how cells respond to fluctuating extracellular signals and changing nutrient availability. While transcriptional regulation is one facet of this control, there is a growing appreciation for the importance of protein trafficking in controlling the activity of plasma membrane proteins. In mammalian cells, endocytosis of cytokine and growth factor receptors occurs following binding of the ligand to the receptor (1). Ligand binding triggers the movement of these receptors into clathrin-coated pits, internaliza-tion into endosomes, and subsequent delivery to lysosomes where they are degraded.
In Saccharomyces cerevisiae, a similar mechanism of regulated endocytosis and degradation has been found for several plasma membrane proteins. For example, binding of the ␣-factor mating pheromone to its receptor triggers endocytosis of the ligand-receptor complex and subsequent degradation in the vacuole, the lysosome-like compartment of the yeast cell (2). This response attenuates the effects of mating pheromone during periods of prolonged exposure. Several nutrient transporters are endocytosed in response to the increased availability of a preferred substrate. Glucose, the preferred carbon source for yeast, signals the endocytosis of the GAL2 galactose permease and the MAL61 maltose permease (3)(4)(5)(6). Likewise, NH 4 ϩ signals endocytosis of the GAP1 amino acid permease because ammonium is preferred over amino acids as a source of nitrogen (7,8). Regulated endocytosis also controls the activity of nutrient transporters in response to changing metabolic demands. The FUR4 uracil transporter is endocytosed in nutrient-starved cells or in cells in which protein synthesis is inhibited (9). This decrease in uracil uptake capacity may prevent futile RNA synthesis. Finally, yeast transporters can be removed from the plasma membrane in response to high levels of their corresponding substrate; this regulation presumably prevents overaccumulation of that substrate if it suddenly becomes abundant in the cell's environment. For example, the ITR1 inositol transporter is endocytosed in response to high levels of inositol (10).
These examples illustrate the important role that regulated endocytosis of plasma membrane proteins plays in controlling nutrient uptake and responses to extracellular signals. Because of its facile genetics, yeast is an excellent model system for study of this intriguing problem of protein trafficking. In this report, we demonstrate the role of regulated endocytosis in the homeostatic control of a metal ion, zinc. Zinc is an essential nutrient for all organisms. This metal is a catalytic component of over 300 enzymes (11) and also plays a structural role in many proteins. For example, several motifs found in transcriptional regulatory proteins are stabilized by zinc including the zinc finger, zinc cluster, and RING finger domains (12). Proteins containing these motifs are very common; for example, almost 2% of the genes in the S. cerevisiae genome encode proteins with zinc-dependent DNA-binding domains (13,14). Although zinc is an essential nutrient, it can be toxic if excess amounts are accumulated. The precise cause of zinc toxicity is unknown but the metal may bind to inappropriate intracellular ligands or compete with other metals for enzyme active sites, transporter proteins, etc. Therefore, in the face of fluctuating extracellular zinc levels, cells must maintain an adequate intracellular zinc level to meet cellular requirements while preventing metal ion overaccumulation.
Mechanisms of regulating intracellular zinc levels or availabil-ity include binding of the metal by metallothioneins (15), zinc storage in intracellular compartments (16), and transport of the metal out of the cell (17). The primary control point for zinc homeostasis in S. cerevisiae is the regulation of zinc uptake across the plasma membrane. Zinc uptake activity in yeast is mediated by two systems. A high affinity system is active in zinc-limited cells and the ZRT1 gene encodes the transporter for this system (18). A low affinity transporter is active in zincreplete cells and is encoded by the ZRT2 gene (19). ZRT1 and ZRT2 proteins share 67% sequence similarity and share a similarly high degree of relatedness to the IRT1 Fe 2ϩ transporter (20) and the ZIP1, ZIP2, ZIP3, and ZIP4 zinc transporters of Arabidopsis thaliana (21). These proteins are members of a growing family of metal ion transporters that have also been identified in protozoans, nematodes, and humans. Transcription of both ZRT1 and ZRT2 is induced in zinclimited cells, this induction is mediated by the ZAP1 transcriptional activator (22). In low zinc conditions, ZAP1 activates expression of the ZRT1 and ZRT2 genes by as much as 30-fold. Zinc inhibits ZAP1 function and limits expression in zinc-replete cells through an unknown mechanism. In this report, we demonstrate that the ZRT1 zinc transporter is also regulated by a separate, post-translational mechanism. We found that under low zinc conditions, ZRT1 is a stable plasma membrane protein. Exposure of cells to high zinc levels triggers internalization of the protein via endocytosis and its subsequent degradation in the vacuole. This post-translational regulatory system limits zinc uptake when cells are exposed to a sudden increase in extracellular zinc levels, thereby preventing the potentially harmful overaccumulation of the metal.

MATERIALS AND METHODS
Yeast Strains and Growth Conditions-Strains used in this study are described in Table I. DEY1531 and RG3248 are isogenic to DY1457 and were generated by backcrossing the relevant mutant alleles (end4::LEU2 and pep4::HIS3 prb1::LEU2, respectively) from their nonisogenic parent strains into the DY1457 genetic background. Yeast were grown in SD medium (0.67% yeast nitrogen base without amino acids) supplemented with auxotrophic requirements and either 2% glucose or 2% galactose. Where indicated, this medium was made zinc limiting by adding 1 mM EDTA. A low zinc medium (LZM), 1 prepared in a similar manner as LIM (26), had the following composition: 0.17% yeast nitrogen base without amino acids, (NH 4 ) 2 SO 4 , or zinc (BIO101); 0.5% (NH 4 ) 2 SO 4 ; 10 mM trisodium citrate, pH 4.2; 2% glucose; 1 mM Na 2 EDTA; and 1% adenine, histidine, leucine, and tryptophan. The MnCl 2 and FeCl 3 concentrations in LZM were adjusted to final concentrations of 25 and 10 M, respectively. Cell number in liquid cultures was determined by measuring the optical density of the cell suspension at 600 nm (OD 600 ) and converting to cell number with a standard curve.
Plasmid and DNA Manipulations-Escherichia coli and yeast transformations were performed using standard methods (27,28). Plasmids used in this study are described in Table II. An epitope-tagged ZRT1 allele was constructed using overlap extension polymerase chain reaction (29). A 900-base pair SacI-PflMI fragment was generated in which a sequence encoding a single hemagglutinin (HA) epitope (30) was fused to the ZRT1 open reading frame immediately following the methionine initiation codon. This fragment was inserted into SacI-PflMI digested pMC5-HS (18) to generate pMC5-HSET. Plasmid pOE1-ET was constructed by isolating the epitope-tagged ZRT1 open reading frame from pMC5-HSET by polymerase chain reaction using primers containing either a BamHI or a SacI site on their 5Ј ends. This fragment was then inserted into BamHI-SacI-digested pRS316-GAL1 (31). Plasmid pOE1-3ET was constructed by digesting pOE1-ET with AatII, which cuts within the HA epitope sequence, and inserting a double-stranded oligonucleotide encoding two additional HA epitopes. A 2-kilobase XhoI-SacI fragment containing the GAL1 promoter and the ZRT1-3ET open reading frame was transferred from pOE1-3ET to YIp306 (32) to generate YIp306 -3ET. This plasmid was digested with EcoRV prior to transformation into yeast strains to direct integration of the plasmid at the URA3 locus (33).
Zinc Uptake Assays and Atomic Absorption Spectrophotometry-Zinc uptake assays were performed as described previously for iron uptake (34) except that 65 ZnCl 2 (Amersham) and LZM-EDTA were substituted for 59 FeCl 3 and LIM-EDTA, respectively. Cells were incubated for 5 min in LZM-EDTA plus 1 M 65 Zn, collected on glass fiber filters (Schleicher and Schuell), washed with 10 ml of ice-cold SSW (1 mM EDTA, 20 mM trisodium citrate, 1 mM KH 2 PO 4 , 1 mM CaCl 2 , 5 mM MgSO 4 , 1 mM NaCl, pH 4.2); cell associated radioactivity was measured on a Packard Auto-Gamma 5650 ␥-counter. Measurement of total cell-associated zinc levels was performed by atomic absorption spectrophotometry. Cells were harvested, washed twice with an equal volume of distilled deionized water, twice with an equal volume of 1 mM EDTA, resuspended in one-fifth volume 6 M nitric acid, and incubated at 95°C for 24 h. The acid-digested samples were then assayed for zinc content on a Varian Spectra AA-30 atomic absorption spectrophotometer.
Protein and Immunoblot Methods-Cells (100 ml, OD 600 of 1-2) were harvested by centrifuging 5 min at 1000 ϫ g and washed with an equal volume of distilled water. These cells were resuspended in 20 ml 1 M Tris-SO 4 , pH 9.3, 10 mM dithiothreitol, incubated at 30°C for 10 min, collected by centrifugation, washed once in spheroplasting buffer (1.2 M sorbitol, 20 mM KHPO 4 pH 7.4), resuspended in 10 ml spheroplasting buffer plus zymolyase 20T (Seikagaku) (5 mg/g cells), and incubated at 30°C for 30 -45 min. Cells were then washed three times in an equal volume of spheroplasting buffer, resuspended in 10 ml 0.6 M mannitol, 20 mM HEPES-KOH, pH 7.4, plus protease inhibitors (1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM pepstatin A), and disrupted in a Dounce homogenizer. The homogenate was centrifuged at 3000 ϫ g for 5 min at 4°C and the pellet of unbroken cells was discarded. Where indicated, the supernatant was centrifuged 30 min at 123,000 ϫ g at 4°C and the supernatant (soluble) and pellet (particulate/membrane) fractions were collected. Immunoblots were performed as described (35) using a monoclonal antibody specific to the HA epitope tag (12CA5, BABCO). Horseradish peroxidase-conjugated goat anti-mouse or antirabbit secondary antibodies (Pierce) were used and detected by enhanced chemiluminescence (ECL, Amersham). Plasmid p352-HA (36) and antibody 12CA5 were used for detection of the CTR1 protein and pCB1 (37) and a rabbit anti-FET4 antiserum (38) were used for detection of the FET4 protein. Densitometric scanning was performed using a CCD camera and IMAGE 1.44 software (National Institutes of Health). Linearity of ECL signal intensity was confirmed with a control immunoblot of serially diluted protein samples. EndoH and peptide Indirect Immunofluorescence Microscopy-Indirect immunofluorescence microscopy was performed essentially as described by Pringle et al. (39) with the following modifications. Cells were fixed in 10 volumes of cold methanol (Ϫ20°C) for 30 min. Fixed cells were treated with glusulase to remove the cell wall and bound to polylysine-treated glass slides. The cells were blocked in PBS ϩ 1 mg/ml BSA (PBS/BSA) by incubating at room temperature for 2 h and stained with 12CA5 antibody (1:200 dilution in PBS/BSA) at room temperature for 16 h. Following washing of the cells, goat anti-mouse IgG antibody coupled to ALEXA 488 (Molecular Probes) was applied (1:200 dilution in PBS/BSA) and the cells were incubated at 37°C for 1 h. The slides were then washed 10 times in PBS and viewed using epifluorescence and Nomarski optics.

RESULTS
Immunological Detection of the ZRT1 Protein-To allow immunological detection of the ZRT1 protein, the ZRT1 gene was epitope-tagged with either one or three tandem copies of the hemagglutinin antigen (HA) epitope (YPYDVPDYA) at its amino terminus immediately following the methionine initiation codon. These proteins (referred to as ZRT1-ET and ZRT1-3ET, respectively) are functional as demonstrated by (i) their ability to complement a zrt1 mutant for zinc-limited growth and (ii) 65 Zn uptake activity that is indistinguishable from wild type ZRT1 (data not shown). On immunoblots, multiple protein bands of approximately 40 -45 kDa molecular mass were detected in cells expressing the ZRT1-ET allele (Fig. 1A). These bands were not observed in cells expressing the untagged protein. These results demonstrate that the anti-HA antibody is specific for the epitope-tagged ZRT1 protein and does not detect other proteins in yeast. Similar results were obtained with the ZRT1-3ET protein although the immunoblot signal intensity was approximately 20-fold greater than that of ZRT1-ET (data not shown). A slower migrating form of epitope-tagged ZRT1 was also frequently observed on immunoblots (Fig. 1A, arrow). This may be a dimeric ZRT1 complex given that the molecular mass of this band was 80 -90 kDa, i.e. twice the apparent monomeric ZRT1 molecular mass.
Although ZRT1-ET was not detected in total cell homogenates, the protein was detectable in a subcellular fraction in which cellular membranes were enriched (Fig. 1A). This result suggests that ZRT1 is an integral membrane protein; further experiments confirmed this hypothesis. Treatment with detergents (Triton X-100, n-octyl-␤-D-glucopyranoside) released ZRT1 into the soluble fraction whereas agents that can only dissociate peripheral membrane proteins (i.e. 10 mM Na 2 CO 3 at pH 10, NaCl, NaBr, or urea) (40) did not (data not shown).
The multiple ZRT1 bands observed suggested that this protein may be subject to post-translational modification. To test if ZRT1 was N-glycosylated, a particulate/membrane fraction isolated from cells expressing epitope-tagged ZRT1 was treated with the glycosidases EndoH or peptide N-glycosidase F. En-doH cleaves the chitobiose core of high mannose oligosaccharides from N-linked glycoproteins, whereas peptide N-glycosidase F cleaves between the asparagine and the innermost N-acetylglucosamine of the carbohydrate. Either treatment reduced the apparent molecular mass of ZRT1 from the 40 -45-kDa bands to a single band of 31 kDa (Fig. 1B). These data indicate that the size heterogeneity observed for ZRT1 is due to different degrees of N-linked glycosylation. It should be noted ZRT1 size heterogeneity was not always observed (see below) and the reason for this inconsistency is unknown.
Post-translational Control of ZRT1 Uptake Activity by Zinc-Expression of the ZRT1 gene is regulated at the transcriptional level by zinc; ZRT1 mRNA synthesis is induced by zinc limitation and repressed during culturing in zinc-replete media (18). As we show here, ZRT1 activity is also regulated by zinc through a separate post-translational mechanism. When zinclimited cells expressing a high level of ZRT1 were treated with a high concentration of extracellular zinc, a rapid loss of zinc uptake activity occurred that was not observed when cells were maintained in a zinc-limiting medium ( Fig. 2A). The loss of activity upon zinc treatment was preceded by an initial lag period of 10 -20 min. The post-translational nature of the effect of zinc on ZRT1 activity was demonstrated in two ways. First, treatment of cells with an inhibitor of protein synthesis, cycloheximide, did not prevent the loss of uptake activity in zinctreated cells ( Fig. 2A, dashed lines). Second, zinc-induced loss of uptake activity was also observed in zrt1 mutant cells expressing ZRT1 from the zinc-insensitive GAL1 promoter (GAL1p-ZRT1) (Fig. 2B). In this experiment, expression from the GAL1 promoter was repressed at time 0 by the addition of glucose and protein synthesis was blocked with cycloheximide. Similar data were obtained for the GAL1p-ZRT1 fusion in the absence of cycloheximide (data not shown). From these results, we conclude that (i) the ZRT1 zinc transporter is regulated by zinc by a post-translational mechanism that is separate from the previously characterized transcriptional control; (ii) new protein synthesis is not required for the post-translational response to occur; and (iii) expression of ZRT1 from the GAL1 promoter can be used to study this response without the added complexity of the zinc-responsive transcriptional control.
The half-lives (t1 ⁄2 ) of uptake activity loss were estimated from the linear portions of the graphs in Fig. 2 (Table III). With or without cycloheximide treatment, the t1 ⁄2 for loss of zinc uptake activity decreased by greater than 7-fold following zinc addition. Identical results were obtained with a zrt1 mutant expressing ZRT1-ET from a high-copy plasmid (pMC5-HSET). The t1 ⁄2 of uptake activity loss in zinc-treated cells expressing pMC5-HS ARS-CEN  ZRT1  0  18  pMC5-HSET ARS-CEN  ZRT1  1  This study  pOE1  ARS-CEN  GAL1  0  18  pOE1-ET  ARS-CEN  GAL1  1  This study  pOE1-3ET  ARS-CEN  GAL1  3  This study  Ylp306-3ET Integrating  GAL1  3  This  ZRT1 from the GAL1 promoter was reproducibly higher than that observed in cells expressing the transporter from its own promoter; the reason for this difference is not yet known. These t1 ⁄2 values highlight the stability of ZRT1 uptake activity in low zinc conditions and its remarkable rate of loss in the presence of high zinc.
Degradation of the ZRT1 Protein in Response to Zinc-Immunoblots indicated that zinc-dependent inactivation of ZRT1 activity is accompanied by degradation of the ZRT1 protein.
Following cycloheximide treatment, the ZRT1 protein was stable in zinc-limited cells but rapidly degraded in zinc-treated cells (Fig. 3A). As was the case for the loss of uptake activity, protein loss occurred after an initial lag period of approximately 30 min. Densitometric quantitation of the band intensities in Fig. 3A is shown in Fig. 3B, the t1 ⁄2 for protein loss was determined from these data. In zinc-treated cells, the ZRT1 protein was degraded with a t1 ⁄2 39 min) that was 25% or less of the t1 ⁄2 (Ͼ150 min) observed in zinc-limited cells. We noted that the loss of protein in zinc-treated cells occurred more slowly than the loss of uptake activity (i.e. compare a t1 ⁄2 for protein of 39 min and a t1 ⁄2 for uptake of 21 min). This result suggested that inactivation of ZRT1 uptake activity is not the direct result of proteolytic degradation but rather that degradation may occur later in the inactivation process. Similar experiments performed with expression of ZRT1 from the GAL1 promoter yielded qualitatively similar results in which ZRT1 was less stable in zinc-treated cells (Fig. 3C). As was observed for loss of uptake activity, degradation of ZRT1 occurred more slowly (t1 ⁄2 of approximately 65 min) in the cells grown in galactose than in glucose-grown cells.
FIG. 2. Post-translational inactivation of ZRT1 zinc uptake activity in response to zinc. A, wild type cells (DY1457) were grown to exponential phase in a zinc-limiting medium (LZM ϩ 10 M ZnCl 2 ), harvested, and reinoculated into SD glucose medium supplemented with either 2 mM ZnCl 2 (filled symbols) or 1 mM EDTA (open symbols) and in the presence (solid lines) or absence (dashed lines) of cycloheximide (100 g/ml). Cells were harvested at the indicated times and assayed for 65 Zn uptake activity. B, a zrt1 mutant strain (ZHY1) expressing ZRT1-ET from the GAL1 promoter (pOE1-ET) was grown to exponential phase in SD galactose medium, harvested, and reinoculated into SD glucose medium supplemented with 100 g/ml cycloheximide and either 2 mM ZnCl 2 (filled symbols) or 1 mM EDTA (open symbols). Cells were incubated at 30°C, harvested at the indicated times, and assayed for 65 Zn uptake activity. An experiment representative of several repetitions is shown. Each value is the mean of three independent assays and the standard deviations were consistently Ͻ10% of the corresponding mean.

TABLE III
Half-life of zinc uptake activity loss The t 1/2 values (in min) were calculated from the linear portions of the graphs described in the legend to Fig. 2 and are representative of several repetitions of these experiments. Plasmids pMC5-HSET and pOE1-ET are described in Table II

FIG. 3. Inactivation of uptake activity is accompanied by degradation of the ZRT1 protein.
A, a zrt1 mutant strain (ZHY1) expressing the epitope-tagged ZRT1 protein from the ZRT1 promoter (pMC5-HSET) was grown to exponential phase in a zinc-limiting medium (LZM ϩ 10 M ZnCl 2 ), harvested, and reinoculated into SD glucose medium containing cycloheximide (100 g/ml) and either 2 mM ZnCl 2 (ϩZn) or 1 mM EDTA (ϪZn). Cells were incubated at 30°C, harvested at the indicated times, and particulate/membrane fractions were prepared for immunoblot analysis. B, signal intensities of the bands in panel A were measured by densitometry and are plotted as the percent of the t 0 sample. C, effects of zinc on ZRT1 protein level when expressed from the GAL1 promoter and on the levels of other plasma membrane proteins. A zrt1 mutant (ZHY1) expressing ZRT1-3ET from the GAL1 promoter (YIp306 -3ET) and wild type cells (DY1457) expressing either epitope-tagged CTR1 (p352-HA) or FET4 (pCB1) were grown to exponential phase in SD galactose medium (CTR1 expression was induced by the addition of 1 mM EDTA). The cells were harvested and reinoculated into SD glucose medium supplemented with 100 g/ml cycloheximide and either 2 mM ZnCl 2 (ϩZn) or 1 mM EDTA (ϪZn). Cells were then incubated at 30°C, harvested at the indicated times, and total homogenates (ZRT1) or particulate/membrane fractions (CTR1 and FET4) were prepared for immunoblot analysis. The two FET4 bands detected here have been observed previously (38).
To determine if the degradation of ZRT1 in response to zinc was specific to ZRT1 or part of a more global change in plasma membrane protein composition, we examined the stability of other yeast plasma membrane proteins in zinc-treated cells. The abundance of the CTR1 copper transporter, the FET4 iron transporter, and the PMA1 H ϩ -ATPase was not greatly altered by zinc treatment (Fig. 3C, data not shown). Thus, zinc-induced ZRT1 inactivation appears to be a specific response of the zinc transporter.
Specificity of ZRT1 Inactivation for Zinc-The rapid decline of ZRT1 zinc uptake activity is accompanied by an equally dramatic increase in cell-associated zinc levels (Fig. 4A). The maximum cell-associated zinc level (ϳ1.3 nmol/10 6 cells), observed in this experiment after 90 min, was 6-fold higher than the maximum steady-state level observed in our previous studies of zinc-responsive transcriptional control (ϳ0.2 nmol/10 6 cells) (18). Given this extremely high level of metal ion accumulated by zinc-treated cells, it was important to determine if ZRT1 inactivation and degradation was an indirect effect of metal-induced stress or toxicity. Several experiments confirmed that ZRT1 inactivation is indeed a mechanism of zinc homeostasis. First, there was no loss of cell viability caused by any of the treatments described in Figs. 2-4 (data not shown). Second, loss of zinc uptake activity was not triggered by any of several conditions that are known to cause inactivation of other yeast plasma membrane transporters, i.e. deprivation of carbon source, nitrogen, or phosphate, heat treatment, or cycloheximide treatment (5,9) (Fig. 2, data not shown). Third, inactivation was triggered by much lower levels of zinc than the 2 mM concentration used in our initial experiments. Treatment of cells with as little as 1 M zinc resulted in a significant loss of uptake activity and ZRT1 protein (Fig. 4B). Higher levels of zinc caused more dramatic effects on both ZRT1 activity and protein level and was correlated with higher cell-associated zinc levels; these results indicate that the degree of the response can be proportional to the zinc level over a certain range of metal ion concentrations.
To determine the specificity of ZRT1 inactivation for zinc over other metals, cells expressing ZRT1 from the GAL1 promoter were treated for 4 h with 0, 1, 10, or 100 M concentrations of several other metal ions. MnCl 2 and FeCl 3 had no effect on ZRT1 uptake activity, and NiCl 2 caused only a slight decrease at the 10 or 100 M concentrations (Fig. 5). CuCl 2 , CdCl 2 , and CoCl 2 treatments greatly decreased zinc uptake activity albeit only at higher concentrations and/or to lesser degrees than was observed for ZnCl 2 . The decrease in uptake activity in response to copper correlated closely with loss of cell viability (data not shown) but no loss of viability was observed in the cadmium-or cobalt-treated cultures. Studies described below demonstrated that ZRT1 inactivation in response to zinc requires a functional endocytic pathway; ZRT1 inactivation is defective in strains bearing mutations (e.g. end4) that inhibit endocytosis. Inactivation of zinc uptake activity by Cd 2ϩ and Co 2ϩ was inhibited in an end4 mutant strain (data not shown) suggesting that ZRT1 inactivation is also triggered by Cd 2ϩ and Co 2ϩ ions.
Zinc-induced ZRT1 Inactivation Is Inhibited in Endocytosisdefective Mutants-The turnover of many yeast plasma membrane proteins occurs through endocytosis and subsequent vacuolar degradation. In response to a particular signal, these proteins accumulate in clathrin-coated pits and are engulfed FIG. 4. Relationship between cell-associated zinc levels, zinc uptake activity, and ZRT1 protein level. A, a zrt1 mutant (ZHY1) expressing ZRT1-3ET from the GAL1 promoter (YIp306 -3ET) was grown to exponential phase in SD galactose medium. The cells were harvested and resuspended in SD glucose medium supplemented with 100 g/ml cycloheximide and either 2 mM ZnCl 2 (filled symbols) or 1 mM EDTA (open symbols). At the indicated times, cells were harvested and assayed for 65 Zn uptake activity (squares) or cell-associated zinc levels (circles). B, a similar experiment to the one described in panel A was performed except that aliquots of cells were treated with 1 mM EDTA or with 1, 10, 100, or 1000 M ZnCl 2 for 4 h prior to harvest. Total cell homogenates were also prepared for immunoblot analysis. An experiment representative of several repetitions is shown. Each value is the mean of three independent assays and the error bars indicate Ϯ 1 S.D. into newly formed endosomes. The proteins are then transferred to the late endosome and delivered to the vacuole where they are degraded by vacuolar proteases. To assess if this pathway is responsible for zinc-induced ZRT1 inactivation, we first examined the effects of zinc treatment on ZRT1 zinc uptake activity in yeast mutants defective for the initial internalization step of endocytosis. Several genes have been shown to be important for this step to occur efficiently (for review, see Ref. 41), including CHC1, END3, END4, and SAC6. CHC1 encodes the clathrin heavy chain protein; END3 and END4 encode proteins of unknown function; and SAC6 encodes fimbrin, an actin filament bundling protein. Initial zinc uptake activity measured in each of these endocytosis mutants was similar to its isogenic wild type parent, demonstrating that zinc uptake by ZRT1 does not require endocytosis (Fig. 6). Upon zinc treatment, inactivation of ZRT1 zinc uptake occurred more slowly in each of the endocytosis-defective mutants relative to its isogenic wild type parent. These data suggest that the loss of uptake activity observed in zinc-treated cells is the direct result of endocytosis of the ZRT1 protein.
To assess the role of the vacuolar proteases in the degradation of ZRT1, we examined the effects of mutations that eliminate the activity of the two major vacuolar endoproteinases, proteinase A encoded by the PEP4 gene and proteinase B encoded by the PRB1 gene. These two proteases are also required for activity of other hydrolases in the vacuole including carboxypeptidase Y and aminopeptidase I (42). Inactivation of ZRT1 uptake activity by zinc treatment was not altered in the pep4 prb1 mutant but degradation was greatly inhibited (Fig.  7). These results indicate that vacuolar proteases are required for degradation of ZRT1 to occur in response to zinc but are not required for the loss of uptake activity.
Effects of Endocytosis-and Protease-defective Mutations on ZRT1 Localization-The experiments described above suggest that zinc-induced inactivation of ZRT1 activity involves endocytosis of the transporter and its subsequent delivery to the vacuole for degradation. To test this hypothesis directly, immunofluorescence microscopy was used to determine the location of the ZRT1 protein in wild type, end4, and pep4 prb1 mutants with and without zinc treatment (Fig. 8). Little fluorescence was detected in cells expressing the wild type ZRT1 protein (data not shown). In wild type cells expressing the epitope-tagged ZRT1-3ET protein, a bright rim of fluorescence at the cell periphery was seen consistent with a plasma membrane localization of the protein. Plasma membrane ZRT1 staining was not affected by a 4-h incubation in zinc-limiting conditions but completely disappeared following a 4-h incubation in 2 mM zinc. In the end4 mutant, plasma membrane staining was also detected but was unaltered by zinc treatment. In the pep4 prb1 mutant, both plasma membrane and the vacuolar staining was observed prior to zinc treatment; this pattern was also observed following a 4-h incubation in zinclimiting medium. In zinc-treated cells, plasma membrane staining was no longer apparent but vacuolar staining was still observed.
Finally, vesicular intermediates of endocytosis can be trapped in the cytoplasm by incubation at 15°C (43). At this temperature, plasma membrane internalization occurs normally but vesicle cargo accumulates in endosome-like compartments. These endosomal structures have been detected as intermediates in the endocytosis of other yeast plasma membrane proteins (43,44). To see if ZRT1 also passes through this intermediate compartment en route to the vacuole, we determined the localization of ZRT1 in cells incubated at 15°C with and without zinc treatment. Incubation at 15°C for 4 h without zinc treatment did not alter zinc uptake activity (data not shown) nor did it alter the plasma membrane localization of ZRT1 (Fig. 8). Inactivation of uptake activity occurred normally FIG. 7. Mutants defective in vacuolar proteases are defective for ZRT1 degradation. Wild type (DY1457, solid lines) and mutant cells (RG3248, dashed lines) expressing ZRT1-3ET from the GAL1 promoter (YIp306 -3ET) were grown to exponential phase in SD galactose medium. The cells were then harvested and resuspended in SD glucose medium supplemented with 100 g/ml cycloheximide and either 2 mM ZnCl 2 (filled symbols) or 1 mM EDTA (open symbols). At the indicated times, cells were harvested, and assayed for 65 Zn uptake activity. Each value is the mean of three independent assays and the error bars indicate Ϯ 1 S.D. B, total cell homogenates were also prepared from zinc-treated cells for immunoblot analysis. FIG. 6. Endocytosis-defective mutants are defective for zincinduced ZRT1 inactivation. Wild type (solid lines) or mutant (dashed lines) cells of the indicated genotype were grown to exponential phase under conditions that induced expression of ZRT1 (SD glucose ϩ 1 mM EDTA at 24°C for panels A and B, SD galactose at 30°C for panels C and D). The cells were then harvested and resuspended in SD glucose medium supplemented with 100 g/ml cycloheximide and either 2 mM ZnCl 2 (filled symbols) or 1 mM EDTA (open symbols) at 37°C for the temperature-sensitive chc1 ts and end3 ts mutants (panels A and B) or 30°C (panels C and D). At the indicated times, cells were harvested and assayed for 65 Zn uptake activity. Because these mutations were generated in different strains than our standard wild type DY1457 strain, the results obtained with the corresponding isogenic parent are also shown. A, CHC1 (GPY1100␣) and chc1 ts (GPY418)) transformed with pMC5-HSET. B, END3 (RH144 -3D) and end3 ts (RH266 -1D) transformed with pMC5-HSET. C, END4 (DY1457) and end4 (DEY1531) transformed with YIp306 -3ET. D, SAC6 (AAY1046) and sac6 (AAY1048) transformed with YIp306 -3ET. An experiment representative of several repetitions is shown. Each value is the mean of three independent assays and the error bars indicate Ϯ1 S.D.
in zinc-treated cells incubated at 15°C (data not shown), consistent with endocytosis being unaffected at this temperature. However, these zinc-treated cells accumulated ZRT1 protein in cytoplasmic bodies similar to the endosome-like structures observed in previous studies. Taken together, these results demonstrate that ZRT1 inactivation results from removal of the ZRT1 protein from the plasma membrane by endocytosis. The subsequent degradation of the protein is a consequence of its delivery to the vacuole. Vacuolar staining observed in the pep4 prb1 mutant in the absence of zinc treatment further suggests that ZRT1 is endocytosed to the vacuole even in the absence of zinc treatment, although at a slower rate than in zinc-treated cells. DISCUSSION Previous studies demonstrated that zinc uptake in yeast is regulated at the transcriptional level by intracellular zinc lev-els (18,22). A zinc-responsive transcriptional activator protein called ZAP1 induces expression of the ZRT1 gene when cells are grown under zinc-limiting conditions. Zinc represses the activation function of ZAP1 through an unknown mechanism. In this report, we describe a second mechanism that controls zinc accumulation in yeast at a post-translational level; zinc induces the endocytosis of the ZRT1 transporter and its subsequent vacuolar degradation.
The post-translational ZRT1 regulatory mechanism is clearly separate from the transcriptional control system given that inactivation of ZRT1 uptake activity occurs normally in a zap1 deletion mutant. 2 However, these two systems undoubtedly work together to maintain the homeostatic control of intracellular zinc levels. It is interesting to note that the transcriptional control system exerts its greatest effect on ZRT1 expression when cell-associated zinc levels vary between 0.01 and 0.07 nmol of Zn/10 6 cells (i.e. ϳ0.5-4 ϫ 10 7 atoms zinc/cell). Approximately 90% repression of a ZRT1-lacZ fusion was observed when cell-associated zinc levels rose to 0.07 nmol/10 6 cells. 3 In contrast, the post-translational response is triggered only at cell-associated zinc levels of greater than 0.07 nmol of Zn/10 6 cells (Fig. 4). Thus, we envision a two-tiered regulatory system in which the transcriptional control can respond to moderate changes in zinc availability and the post-translational control responds to more extreme variations. A likely scenario in which the post-translational control would be important for maintaining zinc homeostasis is when zinc-limited cells are suddenly exposed to high levels of zinc. The rapid down-regulation of zinc uptake by ZRT1 endocytosis helps to prevent overaccumulation of zinc and this would not be possible solely through the transcriptional control of a stable plasma membrane protein. During inactivation of zinc uptake activity, other systems may be induced to facilitate storage of the excess zinc or mediate its efflux from the cell.
The model that we propose for inactivation of the ZRT1 zinc transporter is as follows. In cells in which the intracellular zinc levels are low-to-moderate (i.e. Ͻ0.07 nmol/10 6 cells), the ZRT1 transporter is a stable plasma membrane protein and its level is controlled by the transcriptional regulatory system. When intracellular zinc levels rise above this level, the ZRT1 protein is recruited into clathrin-coated pits as they form at the plasma membrane and then internalized as the endosome pinches off into the cytoplasm. The ZRT1-containing endosomes fuse with the late endosome where the transporter is packaged into vesicles for delivery to the vacuole. Upon arriving in the vacuole, proteases found in this organelle degrade the transporter. An alternative model of the post-translational regulation of ZRT1 is that the transporter is a constitutively recycling protein. In this scenario, ZRT1 is endocytosed at a constant rate and transferred to the endosome. Under low zinc conditions, the protein is returned to the plasma membrane with no net loss of uptake activity. When cells are exposed to high zinc, however, ZRT1 would be routed instead to the late endosome and then to the vacuole for degradation. We think that this model is unlikely. If ZRT1 was a constitutively recycling protein and was being rapidly endocytosed in the absence of zinc treatment, incubating cells at 15°C would cause the protein to accumulate in the endosomes and ultimately deplete the transporter from the plasma membrane. However, zinc uptake activity did not decrease when cells were incubated at 15°C in the absence of zinc treatment. Thus, we found no evidence for constitutive recycling of the ZRT1 protein between the plasma membrane and intracellular endosomal compartments. 2 H. Luo and D. Eide, unpublished result. 3 Zhao, H., Butler, E., Rodgers, J., Spizzo, T., Duesterhoeft, S., and Eide, D. (1998) J. Biol. Chem. 273, in press. FIG. 8. Effect of end4 and pep4 prb1 mutations on ZRT1 localization and abundance. Wild type (DY1457), end4 (DEY1531), and pep4 prb1 (RG3248) strains expressing ZRT1-3ET from the GAL1 promoter (YIp306 -3ET) were grown to exponential phase in SD galactose medium. The cells were then harvested and resuspended in SD glucose medium supplemented with 100 g/ml cycloheximide and either 2 mM ZnCl 2 (ϩZn) or 1 mM EDTA (ϪZn) and incubated at 30°C (or at 15°C as indicated). At 0 (t 0 ) and 4 h after reinoculation, cells were harvested, fixed, stained for ZRT1 protein, and viewed by epifluorescence and Nomarski optics. The intracellular indentations observed in the Nomarski images correspond to the vacuole.
The zinc-induced endocytosis model that we favor raises a number of exciting new questions. First, while it is clear that zinc induces endocytosis of ZRT1, it is unknown if this response is induced by a mechanism that senses intracellular or extracellular metal ion levels. Second, it is unclear if the signal being monitored is Zn 2ϩ ions per se, the activity of a zinc-dependent or zinc-inhibited enzyme, or a more indirect consequence of high metal accumulation. The observation that Co 2ϩ and Cd 2ϩ may also induce endocytosis of ZRT1 is potentially instructive. Both Co 2ϩ and Cd 2ϩ have similar coordination chemistries to Zn 2ϩ and will bind to protein ligands in a similar fashion but generally cannot replace zinc as a functional enzymatic cofactor. Therefore, the simplest hypothesis is that Zn 2ϩ ions trigger endocytosis directly and that Co 2ϩ and Cd 2ϩ mimic that signal. The lower activity of Co 2ϩ and Cd 2ϩ in triggering the response may be due to a greater specificity of the sensing mechanism for Zn 2ϩ , different uptake efficiencies for different metal ions, or some other factor. A third unanswered question is how the zinc signal is transmitted to ZRT1. This could occur through the metal binding directly to the transporter or through an indirect signal transduction pathway. We noted previously the presence of a potential metal-binding site in a presumptive cytoplasmic domain of ZRT1 (18); Zn 2ϩ binding to this site could induce endocytosis of the protein.
How could zinc alter the rate of internalization of the transporter protein? Internalization of many plasma membrane proteins has been shown to be dependent on small, discrete sequences called "internalization signals" that are located in cytoplasmic regions of the proteins (46). These signals include the "tyrosine-based" signal (with the consensus NPXY or YXXZ, where X is any amino acid and Z is a bulky hydrophobic amino acid) and the "dileucine-based" single, i.e. a pair of leucines or other bulky hydrophobic amino acids. Several such elements can be identified in the ZRT1 amino acid sequence. Studies have shown that functional internalization signals bind directly to the AP-2 adaptin complex of clathrin-coated pits and this binding event mediates recruitment of plasma membrane proteins to these structures for internalization (47,48). In the case of ZRT1, we propose that zinc increases the activity of an internalization signal in a cytoplasmic domain of ZRT1 and thereby increases its rate of endocytosis. The activity of an internalization signal in ZRT1 could be modulated by direct binding of zinc to the transporter, interaction of ZRT1 with other proteins, or covalent modification of the ZRT1 protein. Ubiquitination is required for efficient endocytosis of several yeast proteins (2,6,49) and may be involved in ZRT1 endocytosis as well.
Transcriptional regulation has long been known as an important component of metal ion homeostasis. Regulated protein trafficking is now being recognized as an equally important mechanism for controlling the activity and/or function of metal ion transporters. Two examples of this regulation come from recent studies of eukaryotic copper transporters. In cultured mammalian cells, the Menkes copper-transporting P-type AT-Pases is localized to the trans-Golgi network where it supplies the metal to secreted copper-dependent enzymes (45). Treating these cells with copper causes the protein to relocalize to the plasma membrane, presumably to facilitate direct copper efflux from the cell. In yeast, the CTR1 copper transporter is endocytosed in response to copper in a manner analogous to what we have observed for ZRT1 and zinc (44). Although copper uptake was not measured in that study, CTR1 internalization probably facilitates down-regulation of copper uptake activity and thereby limits copper accumulation. Copper was also found to induce the proteolytic degradation of CTR1. In contrast to ZRT1, however, CTR1 degradation did not require vacuolar proteases nor did it require endocytosis; CTR1 degradation in response to high copper appears to occur at the plasma membrane. These examples and our studies of zinc-induced ZRT1 inactivation highlight the importance of regulated protein trafficking in metal ion homeostasis.