Biochemical Properties of Vacuolar Zinc Transport Systems ofSaccharomyces cerevisiae
2002; Elsevier BV; Volume: 277; Issue: 42 Linguagem: Inglês
10.1074/jbc.m205052200
ISSN1083-351X
AutoresColin W. MacDiarmid, Mark A. Milanick, David Eide,
Tópico(s)Plant Micronutrient Interactions and Effects
ResumoThe yeast vacuole plays an important role in zinc homeostasis by storing zinc for later use under deficient conditions, sequestering excess zinc for its detoxification, and buffering rapid changes in intracellular zinc levels. The mechanisms involved in vacuolar zinc sequestration are only poorly characterized. Here we describe the properties of zinc transport systems in yeast vacuolar membrane vesicles. The major zinc transport activities in these vesicles were ATP-dependent, requiring a H+ gradient generated by the V-ATPase for function. One system we identified was dependent on the ZRC1 gene, which encodes a member of the cation diffusion facilitator family of metal transporters. These data are consistent with the proposed role of Zrc1 as a vacuolar zinc transporter. Zrc1-independent activity was also observed that was not dependent on the closely related vacuolar Cot1 protein. Both Zrc1-dependent and independent activities showed a high specificity for Zn2+over other physiologically relevant substrates such as Ca2+, Fe2+, and Mn2+. Moreover, these systems had high affinities for zinc with apparentK m values in the 100–200 nm range. These results provide biochemical insight into the important role of Zrc1 and related proteins in eukaryotic zinc homeostasis. The yeast vacuole plays an important role in zinc homeostasis by storing zinc for later use under deficient conditions, sequestering excess zinc for its detoxification, and buffering rapid changes in intracellular zinc levels. The mechanisms involved in vacuolar zinc sequestration are only poorly characterized. Here we describe the properties of zinc transport systems in yeast vacuolar membrane vesicles. The major zinc transport activities in these vesicles were ATP-dependent, requiring a H+ gradient generated by the V-ATPase for function. One system we identified was dependent on the ZRC1 gene, which encodes a member of the cation diffusion facilitator family of metal transporters. These data are consistent with the proposed role of Zrc1 as a vacuolar zinc transporter. Zrc1-independent activity was also observed that was not dependent on the closely related vacuolar Cot1 protein. Both Zrc1-dependent and independent activities showed a high specificity for Zn2+over other physiologically relevant substrates such as Ca2+, Fe2+, and Mn2+. Moreover, these systems had high affinities for zinc with apparentK m values in the 100–200 nm range. These results provide biochemical insight into the important role of Zrc1 and related proteins in eukaryotic zinc homeostasis. cation diffusion facilitator hemagglutinin 4-morpholineethanesulfonic acid 9-amino-6-chloro-2-methoxyacridine adenosine 5′-(β,γ-imino)triphosphate carbonyl cyanidep-trifluoromethoxyphenylhydrazone 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid Zinc is an essential catalytic component of over 300 enzymes and a critical component of structural motifs such as zinc fingers. Zinc is estimated to be required for the function of more than 3% of the yeast proteome (1Eide D.J. Annu. Rev. Nutr. 1998; 18: 441-469Crossref PubMed Scopus (244) Google Scholar), and therefore the cells must possess mechanisms to obtain sufficient quantities of this important nutrient. Although zinc is not redox-active under physiological conditions, excess zinc can be toxic to cells. Zinc toxicity may be mediated via binding of the cation to inappropriate sites in proteins or co-factors. For example, excess zinc can interfere with mitochondrial aconitase activity and impair respiration (2Costello L.C. Liu Y. Franklin R.B. Kennedy M.C. J. Biol. Chem. 1997; 272: 28875-28881Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). The essential but potentially toxic nature of zinc necessitates precise homeostatic control mechanisms. InEscherichia coli, zinc homeostasis is accomplished largely through the transcriptional control of zinc uptake and efflux transporters (3Patzer S.I. Hantke K. Mol. Microbiol. 1998; 28: 1199-1210Crossref PubMed Scopus (390) Google Scholar, 4Brocklehurst K.R. Hobman J.L. Lawley B. Blank L. Marshall S.J. Brown N.L. Morby A.P. Mol. Microbiol. 1999; 31: 893-902Crossref PubMed Scopus (210) Google Scholar). Recent studies of the regulatory zinc sensors that control expression of these transporters suggest that E. coli cells strive to maintain essentially no labile zinc in their cytoplasm under steady state growth conditions (5Outten C.E. O'Halloran T.V. Science. 2001; 292: 2488-2492Crossref PubMed Scopus (1173) Google Scholar). Similarly, in eukaryotic cells, labile zinc levels are estimated to be in the low nanomolar range under steady state conditions (6Sensi S.L. Canzoniero L.M., Yu, S.P. Ying H.S. Koh J.Y. Kerchner G.A. Choi D.W. J. Neurosci. 1997; 17: 9554-9564Crossref PubMed Google Scholar). Studies of the yeast Saccharomyces cerevisiae have uncovered many aspects of zinc homeostasis in this eukaryotic organism. One major component is the transcriptional regulation of the Zrt1 and Zrt2 zinc uptake transporters. Expression of these proteins is induced under zinc-limiting conditions by the Zap1 transcriptional activator (7Zhao H. Eide D.J. Mol. Cell. Biol. 1997; 17: 5044-5052Crossref PubMed Scopus (223) Google Scholar). A second important mechanism of zinc homeostasis is the post-translational inactivation of Zrt1 by high zinc. When zinc-limited cells are transferred to zinc-replete conditions, zinc rapidly accumulates in cells because of the high level expression of the plasma membrane transporters. We refer to this condition as "zinc shock." In response to zinc shock, Zrt1 is ubiquitinated and removed from the plasma membrane by endocytosis (8Gitan R.S. Lou H. Rodgers J. Broderius M. Eide D. J. Biol. Chem. 1998; 273: 28617-28624Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 9Gitan R.S. Eide D.J. Biochem. J. 2000; 346: 329-336Crossref PubMed Scopus (147) Google Scholar). This results in a rapid decrease in zinc uptake activity that helps limit zinc overaccumulation under these conditions. Yet another critical mechanism of zinc homeostasis is the sequestration of zinc in the vacuole. Several studies suggest that the vacuole is a storage/detoxification site for excess zinc under steady state high zinc conditions. First, mutations that disrupt vacuolar biogenesis or activity of the V-ATPase, which acidifies the vacuole, are hypersensitive to zinc treatment (10Eide D. Bridgham J.T. Zhong Z. Mattoon J. Mol. Gen. Genet. 1993; 241: 447-456Crossref PubMed Scopus (82) Google Scholar, 11Ramsay L.M. Gadd G.M. FEMS Microbiol. Lett. 1997; 152: 293-298Crossref PubMed Google Scholar). Second, mutations that disrupt vacuolar accumulation of polyphosphate, a potential zinc-binding component in the vacuole, are also zinc-sensitive (12Nelson N. Perzov N. Cohen A. Hagai K. Padler V. Nelson H. J. Exp. Biol. 2000; 203: 89-95Crossref PubMed Google Scholar,13Ogawa N. DeRisi J. Brown P.O. Mol. Biol. Cell. 2000; 11: 4309-4321Crossref PubMed Scopus (416) Google Scholar). Third, several potential zinc transporters, Zrc1, Cot1, and Zrt3, have been implicated in vacuolar zinc sequestration. Zrt3 is a member of the ZIP family, which also includes the Zrt1 and Zrt2 transporters and has been proposed to efflux stored zinc from the vacuole to the cytosol during the transition from high to low zinc (14MacDiarmid C.W. Gaither L.A. Eide D. EMBO J. 2000; 19: 2845-2855Crossref PubMed Scopus (309) Google Scholar). Zrc1 and Cot1 are members of the cation diffusion facilitator (CDF)1 family, a ubiquitous group of transporter proteins found in prokaryotes and eukaryotes (15Nies D.H. Silver S. J. Ind. Microbiol. 1995; 14: 186-199Crossref PubMed Scopus (418) Google Scholar, 16Gaither L.A. Eide D.J. BioMetals. 2001; 14: 251-270Crossref PubMed Scopus (433) Google Scholar, 17Paulsen I.T. Saier M.H. J. Membr. Biol. 1997; 156: 99-103Crossref PubMed Scopus (297) Google Scholar). Many CDF proteins have been implicated in zinc efflux or compartmentalization, and Zrc1 and Cot1 are thought to transport zinc into the vacuole. Both proteins have been localized to the vacuole membrane (18Li L. Kaplan J. J. Biol. Chem. 1998; 273: 22181-22187Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 19Miyabe S. Izawa S. Inoue Y. Biochem. Biophys. Res. Commun. 2001; 282: 79-83Crossref PubMed Scopus (57) Google Scholar), although these observations must be interpreted with some caution because they were obtained using cells that overexpressed these proteins. The overexpression of Zrc1 or Cot1 confers zinc tolerance (20Kamizono A. Nishizawa M. Teranishi Y. Murata K. Kimura A. Mol. Gen. Genet. 1989; 219: 161-167Crossref PubMed Scopus (164) Google Scholar, 21Conklin D.S. Culbertson M.R. Kung C. Mol. Gen. Genet. 1994; 244: 303-311Crossref PubMed Scopus (58) Google Scholar), which in the case of Zrc1 is associated with increased zinc accumulation in a compartment that is not accessible to the Zap1 transcription factor. 2C. W. MacDiarmid and D. Eide, unpublished data. These observations suggest that Zrc1 and Cot1 mediate the sequestration of excess zinc in the vacuole. Consistent with this model, the zrc1 andcot1 mutations are associated with reduced total cellular zinc accumulation and increased zinc sensitivity (14MacDiarmid C.W. Gaither L.A. Eide D. EMBO J. 2000; 19: 2845-2855Crossref PubMed Scopus (309) Google Scholar, 20Kamizono A. Nishizawa M. Teranishi Y. Murata K. Kimura A. Mol. Gen. Genet. 1989; 219: 161-167Crossref PubMed Scopus (164) Google Scholar,22Bloβ T. Clemens S. Nies D.H. Planta. 2002; 214: 783-791Crossref PubMed Scopus (87) Google Scholar).2 Interestingly, although the zinc sensitivity of yeast is significantly increased by the zrc1 mutation, such mutant strains are still tolerant to millimolar concentrations of zinc, which are unlikely to be encountered in the natural environment. Our recent studies suggest that an important physiological role of Zrc1 is to tolerate the rapid influx of zinc that results from exposing zinc-deficient yeast to zinc-replete conditions (zinc shock).2 Despite the transcriptional and post-translational control of plasma membrane zinc transporters, zinc shock still results in the overaccumulation of large quantities of zinc (8Gitan R.S. Lou H. Rodgers J. Broderius M. Eide D. J. Biol. Chem. 1998; 273: 28617-28624Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). TheZRC1 gene is transcriptionally induced by Zap1 in zinc-deficient cells (23Lyons 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, 24Miyabe S. Izawa S. Inoue Y. Biochem. Biophys. Res. Commun. 2000; 276: 879-884Crossref PubMed Scopus (34) Google Scholar), and this up-regulation is required to provide tolerance to zinc shock.2 Mutation of theZRC1 gene renders cells extremely sensitive to zinc shock, as does the disruption of ZRC1 induction by Zap1. Hence, the vacuole appears to play a role in buffering cytoplasmic zinc against changes in zinc availability. To fully understand the role of the vacuole in zinc homeostasis, a biochemical characterization of the zinc transporter activities in this compartment is essential. Two previous studies have addressed this issue. Okorokov et al. (25Okorokov L.A. Kulakovskaya T.V. Lichko L.P. Polorotova E.V. FEBS Lett. 1985; 192: 303-306Crossref PubMed Scopus (40) Google Scholar) were the first to assay zinc transport activity in yeast vacuolar vesicles. These investigators characterized a transport system with an apparent K mof 55–170 μm. However, this apparent low affinity for substrate is unlikely to be of physiological significance given the low amounts of labile zinc (i.e. nm) now thought to be present in cells. Similarly, White and Gadd (26White C. Gadd G.M. J. Gen. Microbiol. 1987; 133: 727-737Google Scholar) noted zinc transport activity in vacuole vesicles, but this activity was not further described. Because of these considerations, we initiated this study of the biochemical properties of zinc transport activities in vacuolar vesicles. Furthermore, using genetic manipulations in combination with our biochemical assays, we investigated the contribution of the CDF family proteins and the V-ATPase to vacuolar zinc transport. Yeast were routinely grown in YPD or in synthetic defined medium with 2% glucose and necessary auxotrophic supplements. LZM was prepared as described previously (8Gitan R.S. Lou H. Rodgers J. Broderius M. Eide D. J. Biol. Chem. 1998; 273: 28617-28624Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 27Zhao H. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2454-2458Crossref PubMed Scopus (453) Google Scholar). Low zinc YPD (LZ-YPD) medium was prepared by treating standard YPD medium (1 liter) with 25 g of Chelex 100 resin for 12 h at 4 °C. The resin was removed, and the medium was supplemented with 4 mm MgCl2, 100 μm CaCl2, 25 μmMnCl2, 10 μm FeCl3, and 2 μm CuCl2. LZ-YPD had a zinc content of less than 1 μm as determined by atomic absorption spectroscopy (data not shown). Sodium citrate (20 mm, pH 4.2) and EDTA (1 mm) were added to further limit zinc bioavailability, and the medium was filter-sterilized. Cell number/ml of yeast suspensions was determined by measuring the optical density at 600 nm (A 600) and comparing with a standard curve.E. coli and yeast transformations were performed using standard methods. Immunoblot analysis and indirect immunofluorescence was performed as described previously (9Gitan R.S. Eide D.J. Biochem. J. 2000; 346: 329-336Crossref PubMed Scopus (147) Google Scholar, 14MacDiarmid C.W. Gaither L.A. Eide D. EMBO J. 2000; 19: 2845-2855Crossref PubMed Scopus (309) Google Scholar). The antibodies were obtained from Molecular Probes (Eugene, OR) and Pierce. Affinity purified rabbit Kex2 antibody was a gift of Steve Nothwehr. The ZRC1 gene was amplified from yeast genomic DNA (DY1457) using oligonucleotides designed to include 1000 bp of the promoter and 500 bp of the terminator regions. The resulting DNA fragment was cloned into theEcoRI and PstI sites of pFL38 (28Bonneaud N. Ozier-Kalogeropoulos O., Li, G.Y. Labouesse M. Minvielle-Sebastia L. Lacroute F. Yeast. 1991; 7: 609-615Crossref PubMed Scopus (501) Google Scholar) to generate YCpZRC1. An epitope-tagged ZRC1 allele was generated by overlap PCR (29Ho 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) adding three copies of the Haemophilus influenzae hemagglutinin (HA) epitope to the C terminus of Zrc1. The PCR product was then inserted into BamHI- andHpaI-digested YCpZRC1 by gap repair to generate YCpZRC1-HA. Yeast strains CM100 (ZRC1 COT1), CM102 (zrc1::HIS3), CM103 (cot1::URA3), and CM104 (zrc1::HIS3 cot1::URA3) were described previously (14MacDiarmid C.W. Gaither L.A. Eide D. EMBO J. 2000; 19: 2845-2855Crossref PubMed Scopus (309) Google Scholar). All of the newly constructed strains were isogenic to CM100. CM142 (ZRC1 −HA) was constructed by transformation of CM104 with the insert of YCpZRC1-HA. Transformants were selected by complementation of the zinc-sensitive phenotype of CM104, and the mutations were verified using PCR. The cot1 mutation was removed by backcrossing to a wild-type strain. Yeast strain MM112 (vph1::LEU2 stv1::LYS2) (30Manolson M.F., Wu, B. Proteau D. Taillon B.E. Roberts B.T. Hoyt M.A. Jones E.W. J. Biol. Chem. 1994; 269: 14064-14074Abstract Full Text PDF PubMed Google Scholar) was a gift of Rob Piper. Yeast vacuoles were prepared using the method of Kakinuma et al. (31Kakinuma Y. Ohsumi Y. Anraku Y. J. Biol. Chem. 1981; 256: 10859-10863Abstract Full Text PDF PubMed Google Scholar) with the following modifications. Yeast cells were grown to late log phase in YPD medium with 4% glucose and then washed once with water and once with buffer A (100 mm KPO4 buffer, pH 7.0, 1.2 msorbitol). The cells were resuspended in three times the pellet volume in buffer A with 10 mm dithiothreitol, 1% glucose, and 50 units zymolyase/ml. The cells were incubated for 1–2 h at 30 degrees with gentle shaking. The spheroplasts were washed twice with buffer A and resuspended in 5 ml of lysis buffer (10 mm MES-Tris, pH 6.9, 0.1 mm MgCl2, 12% Ficoll 400). Most cells lysed after resuspension in this buffer, and cell clumps were broken up by 15 strokes in a Dounce homogenizer. The lysate was then processed as described previously (31Kakinuma Y. Ohsumi Y. Anraku Y. J. Biol. Chem. 1981; 256: 10859-10863Abstract Full Text PDF PubMed Google Scholar), except that membrane wafers were resuspended by repeated pipetting. The final wafer of vacuoles was resuspended in 5 ml of 1× buffer C (10 mm MES-Tris, pH 6.9, 25 mm KCl, 4 mm MgCl2) and dispersed by repeated pipetting. Vesiculated vacuole membranes were collected by centrifugation at 60,000 × g for 30 min, resuspended in buffer C with 10% glycerol, and frozen in liquid nitrogen prior to storage at −80 °C. The protein content of vacuole preparations was assayed using a Bradford assay kit (Bio-Rad). Efforts to reduce the degree of Zrc1 degradation during vesicle preparation using various protease inhibitor mixtures were unsuccessful. Accumulation of65Zn2+ by vacuole vesicles was assayed in 10 mm MES-Tris buffer (pH 6.9) with 4 mmMgCl2 and 25 mm KCl. Vacuole vesicles (routinely 5 μg of protein) were added to 200 μl of this buffer with or without 1 mm ATP and the indicated65Zn2+ concentration and incubated at 30 °C. The reactions were stopped by addition of 4.5 ml of cold wash buffer (10 mm MES-Tris, pH 6.9, 25 mm KCl, 1 mm Tris-buffered EDTA) and incubation on ice. The vesicles were collected on nitrocellulose filters (Millipore; pore size, 0.45 μm) and washed three times with 5 ml of cold wash buffer. The radioactivity retained on the filters was quantified in a γ counter. Michaelis-Menten constants were determined by nonlinear interpolation of the data using Prism (version 3.0a for Macintosh, GraphPad Software, San Diego, CA). Adenosine-triphosphatase activity of isolated vacuole membranes was assayed in 10 mm Tris-MES buffer (pH 6.9) containing 5 mm MgCl2, 25 mm KCl, and 1 mm Mg2+-ATP. Aliquots of 500 μl of buffer were incubated at 30 °C, and vacuole vesicles (5 μg of protein) were added to start the assay. The assay was terminated by addition of 1 ml of stop buffer (2% H2SO4, 0.5% NH4MoO4, 0.5% SDS) and color developed by the addition of 10 μl of freshly prepared 10% sodium ascorbate. After 10 min of color development, the samples were read at 750 nm against a blank with no protein added. Sodium orthovanadate was prepared by boiling a 100 mm stock of sodium vanadate for 10 min and used immediately. To assay changes in the internal pH of vacuole vesicles, vacuole membranes (10 μg of protein) were added to 1 ml of ATPase assay buffer in a spectrofluorimeter cuvette at 30 °C. 9-Amino-6-chloro-2-methoxyacridine (ACMA) was added to a final concentration of 2 μm. Dye fluorescence was excited by light of wavelength 410 nm, and the emission at 482 nm was continuously recorded using a Hitachi F3010 spectrofluorimeter. When a stable emission signal was obtained, ATP (1 mm) was added, and quenching of the dye fluorescence was recorded. Many genetic studies have implicated the importance of vacuolar zinc transport in zinc homeostasis. To examine this process at a biochemical level, vacuolar vesicles were isolated from yeast cells for in vitro zinc transport studies. The purity of these preparations was assessed by immunoblot analysis using antibodies against marker proteins of known compartments. Compared with whole cell lysates, vacuole vesicles from wild-type cells were enriched ∼4-fold in two vacuolar marker proteins, Cpy and Vma1 (Fig.1). The levels of marker proteins for other compartments such as mitochondria, Golgi, and endoplasmic reticulum, as well as a cytoplasmic enzyme, were at similar or reduced levels in the vacuole fraction. These data indicated that the isolation procedure resulted in a significant enrichment of vacuole vesicles. Similar results were obtained with vesicles isolated fromzrc1 mutant cells (Fig. 1). Experiments using vesicles from this mutant strain will be described later in this report. From previous reports of this isolation procedure, it was known that vacuole membrane vesicles were primarily obtained in the right-side-out orientation, making them suitable for studies of vacuolar cation import (31Kakinuma Y. Ohsumi Y. Anraku Y. J. Biol. Chem. 1981; 256: 10859-10863Abstract Full Text PDF PubMed Google Scholar, 32Ohsumi Y. Anraku Y. J. Biol. Chem. 1983; 258: 5614-5617Abstract Full Text PDF PubMed Google Scholar). The observed enrichment of CPY, a lumenal vacuolar protein, indicated that a significant fraction of the vesicles isolated were right-side-out. This conclusion was also supported by our observation that significant V-ATPase activity was detected in nonpermeabilized vesicles (see below). To determine whether vacuole vesicles could accumulate zinc, membranes isolated from a wild-type strain were incubated with radioactive65Zn with or without Mg-ATP as an energy source. Zinc accumulation was assessed over time by collecting the vesicles on nitrocellulose filters, washing them to remove surface-bound zinc, and then measuring the level of accumulated isotope. In the absence of ATP, only a low level of zinc accumulation was observed. In the presence of ATP, however, a high level of zinc uptake activity was detected (Fig.2 A). This ATP-dependent zinc uptake was rapid and essentially complete after 2 min of incubation with 1 μm65Zn. This plateau was likely due to an equilibrium effect (i.e. high lumenal zinc inhibiting additional uptake) because assays using higher concentrations of zinc resulted in longer periods of increasing accumulation and higher final levels of zinc (data not shown). Zinc accumulation was not stimulated by ADP or a nonhydrolyzable analogue of ATP (AMP-PNP), indicating a requirement for hydrolysis of the γ-phosphate of ATP. That the vesicle-associated zinc was actually in the vesicle lumen rather than bound to the membrane surface was confirmed using pyrithione, a zinc ionophore. The addition of pyrithione to vesicles after zinc accumulation caused a rapid decrease in vesicle-associated 65Zn. This effect was not simply due to pyrithione chelating and removing surface-bound zinc, because the membrane-impermeable chelator EDTA was routinely used to wash the vesicles after 65Zn treatment and had no effect on the pyrithione-accessible zinc. These observations indicated that65Zn was accumulated within a membrane-bound compartment. Because pyrithione treatment caused a net release of accumulated zinc, these data also indicated that the vesicles were capable of generating a zinc concentration gradient. The accumulation of ions and metabolites in yeast vacuoles is often mediated by secondary active transport systems, which drive solute transport using the stored energy of a proton concentration gradient generated by the vacuolar V-type H+-ATPase (33Klionsky D.J. Herman P.K. Emr S.D. Microbiol. Rev. 1990; 54: 266-292Crossref PubMed Google Scholar). The vacuole vesicles isolated in this study were capable of generating a proton gradient when supplied with ATP, as determined by quenching of the pH-sensitive fluorescent indicator, ACMA (Fig. 2 B). Generation of this proton gradient was also dependent on a hydrolyzable ATP substrate as was observed for the zinc uptake assays. To determine whether this proton gradient was required for zinc uptake, the effect of compounds that dissipate pH gradients was examined (Table I). As a control, the effect of these inhibitors on total ATPase activity of the extracts and vesicle acidification was also determined. Addition of the protonophore FCCP at the start of the zinc uptake assay severely inhibited this activity. This compound also effectively dissipated the proton gradient generated by adding ATP to ACMA-treated vesicles (Fig.2 B). Consistent with its mode of action, FCCP did not inhibit ATPase activity. In fact, FCCP stimulated this activity, an effect that is consistent with the uncoupling of ATP hydrolysis and vesicle acidification mediated by this inhibitor. Nigericin, an ionophore that dissipates proton gradients via K+/H+ exchange, also inhibited zinc uptake and stimulated ATPase activity. We also tested the effect of valinomycin, a potassium ionophore that dissipates gradients of K+ and electrical potential gradients but does not affect proton gradients. Valinomycin treatment did not inhibit zinc uptake, indicating that the uptake was not dependent on a K+ gradient nor an electrical potential gradient generated by ATPase activity. The above observations indicated that the ATP-dependent accumulation of zinc by vacuole vesicles required a proton concentration gradient. As expected, nigericin dissipated the vesicle proton gradient, whereas valinomycin did not (data not shown).Table IEffect of inhibitors on zinc uptake and ATPase activity of vacuole vesiclesTreatment65Zn uptake rateanmol/mg protein/min.Relative 65Zn uptake ratebPercentage of + ATP value.ATPase specific acitvitycnmol Pi/μg protein/min.Relative ATPase specific activitydPercentage of + ATP value.−ATP0.8 ± 0.25.0 ± 1.0NANA+ATP16.8 ± 0.7100.0 ± 4.21.6 ± 0.1100.0 ± 3.0FCCP4.0 ± 0.723.6 ± 4.04.6 ± 0.1295.2 ± 1.6Nigericin2.4 ± 0.214.5 ± 1.34.1 ± 0.1260.2 ± 8.3Valinomycin17.8 ± 0.3105.7 ± 1.51.6 ± 0.1100.2 ± 2.4Oligomycin13.4 ± 0.980.0 ± 5.01.2 ± 0.175.1 ± 0.4Concanamycin A3.1 ± 0.218.5 ± 1.00.6 ± 0.140.0 ± 2.1Vanadate16.4 ± 0.597.3 ± 3.20.4 ± 0.126.4 ± 3.0Zinc uptake assays were performed as described under "Experimental Procedures" (1-min incubation with 0.5 μm65Zn2+). The reactions contained ATP (1 mm) or ATP and either FCCP (5 μm), nigericin (5 μm), valinomycin (5 μm), oligomycin (10 μg/ml), concanamycin A (100 nm), or sodium vanadate (1 mM). The effect of these inhibitors on total ATPase activity in vacuolar membrane vesicles was also determined. The ATPase assay reactions were incubated for 8 min prior to stopping the reactions. Identical ATP and inhibitor concentrations were used for both zinc uptake and ATPase assays. NA, not applicable.a nmol/mg protein/min.b Percentage of + ATP value.c nmol Pi/μg protein/min.d Percentage of + ATP value. Open table in a new tab Zinc uptake assays were performed as described under "Experimental Procedures" (1-min incubation with 0.5 μm65Zn2+). The reactions contained ATP (1 mm) or ATP and either FCCP (5 μm), nigericin (5 μm), valinomycin (5 μm), oligomycin (10 μg/ml), concanamycin A (100 nm), or sodium vanadate (1 mM). The effect of these inhibitors on total ATPase activity in vacuolar membrane vesicles was also determined. The ATPase assay reactions were incubated for 8 min prior to stopping the reactions. Identical ATP and inhibitor concentrations were used for both zinc uptake and ATPase assays. NA, not applicable. To determine which enzyme was responsible for generating this proton gradient, we tested the effect of inhibitors of the V-type H+-ATPase (concanamycin A), the P-type H+-ATPases (vanadate), and the mitochondrial F1Fo H+-ATPase (oligomycin) on zinc uptake and ATPase activity (Table I). Of the three inhibitors used, concanamycin A most strongly inhibited zinc accumulation. Concanamycin A at a concentration of 100 nm or above maximally inhibited ∼60% of the ATPase activity in yeast vacuole preparations. The remaining concanamycin A-insensitive ATPase activity is likely due to vacuolar phosphohydrolases or from contamination of the preparations with other organelles (Fig. 1). ATP hydrolysis by this concanamycin A-insensitive activity was not coupled to vesicle acidification; 100 nm concanamycin A completely prevented the acidification of vacuole vesicles as determined using ACMA (Fig. 2 B). In contrast, oligomycin and vanadate had far lesser effects on vesicle acidification. Taken together, these data indicate that ATP-dependent zinc accumulation in vacuole vesicles is mediated by secondary active transport systems that are dependent on the proton gradient generated by the V-ATPase. Further evidence for the involvement of the V-ATPase in zinc transport was obtained by assays of vesicles from a vph1 stv1 mutant strain, which lack V-ATPase activity because of loss-of-function mutations in the two V-ATPase 100-kDa subunits (see below). Intracellular glutathione concentrations are in the millimolar range, suggesting that much of the labile cytosolic zinc in cells may be present in the form of zinc-glutathione complexes. Surprisingly, high levels of reduced glutathione (1 mm GSH with 1 μm65Zn, data not shown) did not inhibit ATP-dependent zinc uptake in vitrodespite the ability of glutathione to bind zinc with high affinity (34Krezel A. Wojciech B. Acta Biochim. Pol. 1999; 46: 567-580Crossref PubMed Scopus (101) Google Scholar). These results suggest that zinc-GSH complexes may be a labile source of Zn2+ for transport in vivo. The observation that vacuole vesicles contained zinc transport activity led us to investigate which proteins might be responsible for this activity. Two obvious candidates were the CDF proteins Zrc1 and Cot1; both proteins are likely to be zinc transporters, although direct evidence supporting this hypothesis has not been reported. To determine whether Zrc1 contributes to zinc uptake by vacuole vesicles, we first confirmed the subcellular location of this protein. Zrc1 had previously been localized to the vacuole membrane when overexpressed (18Li L. Kaplan J. J. Biol. Chem. 1998; 273: 22181-22187Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 19Miyabe S. Izawa S. Inoue Y. Biochem. Biophys. Res. Commun. 2001; 282: 79-83Crossref PubMed Scopus (57) Google Scholar), but the overexpression of membrane proteins can lead to their mislocalizatio
Referência(s)