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Originally published In Press as doi:10.1074/jbc.M507481200 on October 31, 2005

J. Biol. Chem., Vol. 281, Issue 1, 410-417, January 6, 2006
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The Yeast Arr4p ATPase Binds the Chloride Transporter Gef1p When Copper Is Available in the Cytosol*{boxs}

Jutta Metz{ddagger}1, Andrea Wächter{ddagger}1, Bastian Schmidt{ddagger}, Janusz M. Bujnicki§2, and Blanche Schwappach{ddagger}23

From the {ddagger}Zentrum für Molekulare Biologie der Universität Heidelberg, Im Neuenheimer Feld 282, Heidelberg D-69120, Germany and the §Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology, Trojdena 4, Warsaw PL-02-109, Poland

Received for publication, July 11, 2005 , and in revised form, October 11, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cellular ion homeostasis involves communication between the cytosol and the luminal compartment of organelles. This is particularly critical for metal ions because of their toxic potential. We have identified the yeast homologue of the prokaryotic ArsA protein, the homodimeric ATPase Arr4p, as a protein that binds to the yeast intracellular CLC chloride-transport protein, Gef1p. We show that binding of Arr4p to the C terminus of Gef1p requires the presence of yeast cytosol and is sensitive to a highly specific copper chelator in vitro and in vivo. Copper alone can substitute for cytosol to support the interaction of Arr4p with the C terminus of Gef1p. The migration behavior of Arr4p in nonreducing gel electrophoresis correlates with cellular copper deficiency, repletion, or stress. Our homology model of Arr4p shows that the antimony (arsenic) metal binding site of ArsA is not conserved in Arr4p. The model suggests that a pair of cysteines, Cys285 and Cys288, is located in the interface of the Arr4p dimer. These residues are required for Arr4p homodimerization and for binding to the C terminus of Gef1p. Whereas both proteins are required for normal growth under iron-limiting conditions, they play opposite roles when copper and heat stress are combined in an alkaline environment. Under these conditions, {Delta}gef1 cells grow much better than wild type yeast, whereas {Delta}arr4 cells are unable to grow. Comparison of the {Delta}arr4 with the {Delta}arr4{Delta}gef1 strain suggests that Arr4p antagonizes the function of Gef1p.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Copper is an essential metal that is toxic when present in higher concentrations. Homeostatic mechanisms keep copper ions available and protect cellular components against its dangerous redox chemistry. In Saccharomyces cerevisiae, key components of copper homeostasis have been identified (e.g. copper-responsive transcription factors, copper transporters, and copper chaperones) (1). The luminal compartment of the late secretory pathway requires copper for the biosynthesis of copper-containing proteins (2). The endosomal system and the vacuole are thought to contribute to copper storage and detoxification (3–5). Little is known about how cytosolic and luminal copper concentrations of the endomembrane system are integrated in cellular copper ion homeostasis.

Gef1p is a CLC chloride channel homologue required for efficient copper supply to the lumen of the Golgi and the prevacuole (6). The recent finding that two of the most closely related mammalian homologues of Gef1p, ClC-4 and -5, exhibit chloride/proton exchange activity (7, 8) like the prokaryotic CLC counterpart (9) suggests that Gef1p may also act as a chloride/proton antiporter. {Delta}gef1 mutants show the following phenotypes: loss of high affinity iron uptake, reduced resistance to toxic cations (e.g. high sodium and hygromycin), and sensitivity to alkaline pH of the growth medium (6, 10–12). High affinity iron uptake is lost because Fet3p (a multicopper oxidase involved in high affinity iron uptake at the cell surface) does not mature normally in {Delta}gef1 strains (6). Copper loading to the active center of Fet3p in the lumen of the late secretory pathway requires Cl- ions that are thought to enter the compartment via Gef1p (13). High external copper can suppress the growth defect at elevated pH (6). This is consistent with the notion that copper and iron are the limiting factors for growth of S. cerevisiae in an alkaline environment (14). In the pathogenic yeast Cryptococcus neoformans, a CLC-type chloride channel gene is required for the activity of the copper-containing enzyme laccase, a major virulence factor (15). One of the most closely related mammalian counterparts of Gef1p, ClC-4, has been shown to stimulate the incorporation of copper into ceruloplasmin in hepatocytes (16).

In contrast to cation channels, almost no accessory proteins involved in CLC protein function and subcellular localization are known. Employing the yeast two-hybrid system, we screened for proteins that interact with the cytosolic C terminus of Gef1p and identified the yeast homologue of the prokaryotic ArsA ATPase, Arr4p, as an interaction partner. Divergent from its function in Escherichia coli, the eukaryotic counterpart of this protein is sensitive to changes in the availability of copper in the cytosol and is required when copper and heat stress are combined.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Yeast Strains and Media—Standard yeast media and genetic manipulations were as described (17). LIM10 and -50 low iron selection media were prepared as described (18). APG medium, a synthetic minimal medium containing 10 mM arginine, 8 mM phosphoric acid, 2% glucose, 2 mM MgSO4, 1 mM KCl, 0.2 mM CaCl2, trace minerals, and vitamins, was prepared and buffered with 10 mM MES and KOH to pH 7.0 as described (19). All strains were derived from K700{alpha} (Mat{alpha}, HML{alpha}, HMB{alpha}, ho, ade2-1, trp1-1, can1-100, leu2-3, 2-112, his3-11, 3-15, ura3, ssd1) (20). The {Delta}gef1 deletion strain has been described (12). {Delta}arr4 deletions were obtained by transforming the strains with a linear DNA fragment containing 200 bp of the 5' and 325 bp of the 3' portion of the ARR4 open reading frame with the TRP1 marker inserted between them. Gene disruption was verified by PCR.

Molecular Biology—Standard molecular biology protocols were adapted from Ref. 17. Epitope-tagged or mutated constructs of GEF1 and ARR4 were created using PCR. All constructs were verified by sequencing and tested for functional complementation of the {Delta}gef1 phenotype on LIM50 medium for GEF1 constructs. The four-protein C (PC)4 epitope was fused to Arr4p using an engineered NotI restriction site, replacing the stop codon of the respective open reading frames with three codons encoding alanine. The sequence of the PC epitope reads EDQVDPRLIDGK. N-terminal HA tagging of Gef1p resulted in the addition of the following sequence to the N terminus of Gef1p: MVGYPYDVPDYAGYPYDVPDYAGST. For the expression of recombinant Arr4p as a maltose-binding protein fusion, the open reading frame of ARR4 was cloned into pMAL-c2X (New England Biolabs). For the expression of recombinant Arr4p as a decahistidine-tagged protein, the open reading frame of ARR4 was cloned into a pQE80 (Qiagen) derivative containing a TEV cleavage site. The portion of the GEF1 gene encoding the CT (codons 572–779) was cloned in frame into a pQE80 derivative containing two Z domains (21).

Yeast Two-hybrid System—For the yeast two-hybrid system, constructs were cloned into vectors pGADT7 and pGBKT7 (BD Biosciences/Clontech). The GEF1 bait construct was transformed into the yeast two-hybrid strain AH109 (BD Biosciences/Clontech), and large scale library transformations of a yeast genomic two-hybrid library (22) in this bait strain were performed according to Ref. 23. Primary co-transformants were screened on synthetic complete (SD) medium lacking tryptophan, leucine, and histidine. Positive colonies were streaked to SD medium lacking tryptophan, leucine, histidine, and adenine and subjected to a {beta}-galactosidase plate assay (overlay of the plates with the following solution containing 0.5% agarose: 0.5 M sodium phosphate (pH 7.0), 0.1% SDS, 2% N,N-dimethylformamide, 0.2% 5-bromo-4-chloro-3-indolyl-{beta}-D-galactopyranoside). Library plasmid DNA from colonies displaying activity of all three reporter genes (HIS3, ADE2, and lacZ) was rescued according to Ref. 17 and retransformed with the original bait construct and a green fluorescent protein-containing control construct. Transformants were tested for the bait-specific activity of the HIS3 and ADE2 reporter genes, and the corresponding plasmids were sequenced. For testing specific yeast two-hybrid constructs, strain AH109 was transformed with the constructs indicated in Figs. 1, 4, and 6 and plated on dropout medium lacking tryptophan and leucine to recover transformants. Individual colonies were grown in liquid culture, and serial dilutions were spotted on dropout medium lacking tryptophan and leucine or lacking tryptophan, leucine, and histidine.

Generation of the Anti-Arr4p Antiserum—Arr4p was expressed in E. coli as a maltose-binding protein fusion. Cleared extracts from lysed bacteria were passed over a column containing amylose resin, and the fusion protein was eluted using maltose according to the manufacturer's protocol (New England Biolabs). Guinea pigs were injected with the purified fusion protein in phosphate-buffered saline (Peptide Specialty Laboratories, Heidelberg, Germany).

Immunoprecipitation and Immunoblotting—Total yeast extracts were prepared from 0.2 A600 units of log phase cells by alkaline lysis under nonreducing conditions and precipitation of total cellular protein by trichloroacetic acid. For co-immunoprecipitation, yeast cells were grown to an A600 of 0.5 in the appropriate dropout media containing 2% glucose as the carbon source. 150 A600 units were harvested and broken in 500 µl of breaking buffer (20 mM HEPES, pH 7.4, 100 mM KCl, 2x Complete protease inhibitor mixture; Roche Applied Science) using glass beads. The resulting extract was diluted 1:1 with immunoprecipitation buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM EDTA, and 1% Nonidet P-40, complemented with Complete protease inhibitor) and incubated with 5 µg of mouse monoclonal anti-HA (HA.11; Covance) or 10 µl of anti-Arr4p antiserum and protein G-Sepharose (Amersham Biosciences) overnight, washed three times in immunoprecipitation buffer and several times in phosphate-buffered saline, and eluted in SDS-PAGE loading buffer without reducing agent at 37 °C. Yeast extracts and immunoprecipitates were separated by SDS-PAGE using 10 or 7% gels and transferred to nitrocellulose. Blots were blocked in TBS (2.7 mM KCl, 137 mM NaCl, 20 mM Tris-HCl, pH 7.5) containing 5% milk powder and 0.02% Nonidet P-40. Primary antibodies (anti-HA mouse monoclonal HA.11 (Covance), 1 µg/ml; anti-PC mouse monoclonal HPC4 (Roche Applied Science), 0.25 µg/ml; anti-Arr4p, 1:2,500) and secondary antibodies (horseradish peroxidase-conjugated anti-mouse and anti-rabbit antibodies (Jackson), 1:4,000) were diluted in TBS-blocking solution. Washes were in TBS-blocking solution and then in TBS, 0.02% Nonidet P-40. Detection was performed using the ECL system (Amersham Biosciences).

Binding Assays Employing Recombinant Proteins—Constructs used as bait in binding assays were expressed in an ompT protease-deficient strain of E. coli and purified on Ni2+-nitrilotriacetic acid-agarose or directly bound to IgG-Sepharose (Amersham Biosciences; 20 µl of slurry/reaction; beads were prewashed in 0.2 M glycine, pH 2.2) in immobilization buffer (50 mM Tris-HCl, pH 7.5, 2 mM Mg(OAc)2, 200 mM NaCl). Beads were washed first in immobilization buffer and then in binding buffer (50 mM Tris-HCl, pH 7.5, 2 mM Mg(OAc)2, 50 mM NaCl). Upon the addition of Arr4p (40 µg/ml), the binding reaction proceeded for 3 h at 4°C in binding buffer or cytosol with further additions as indicated in the text and figure legends. After four washes with binding buffer, bound proteins were eluted with 1.5 M magnesium chloride and precipitated using isopropyl alcohol.

Preparation of Bacterial and Yeast Cytosol—Yeast cultures grown in full medium were harvested in the logarithmic growth phase. The pellets were pressed through a syringe to obtain strings, which were frozen in liquid nitrogen and macerated in a mortar in the presence of liquid nitrogen. The ground material was thawed in an equal volume of binding buffer and centrifuged at 100,000 x g for 2 h. Bacteria were disrupted by sonification, and the lysate was cleared by the same centrifugation step. Fractionation of yeast cytosol by centrifugation through a polyethersulfone membrane was performed according to the manufacturer's instructions (membraPure).

Blue Native Gel Electrophoresis—Blue native PAGE analysis was performed as described by Schägger and von Jagow (24) using bovine serum albumin as a standard.

Inductively Coupled Mass Spectrometry—Trace mineral analysis was carried out by a commercial provider, Spurenanalytisches Laboratorium (Dr. Heinrich Baumann). Arr4p purified from E. coli was dialyzed overnight against 20 mM Hepes, pH 7.5 (titrated with Tris base).

Homology Modeling of Arr4p—The detailed procedure for building the homology model is given as supplemental information.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In search for proteins that would bind to the C terminus (CT) of Gef1p, we screened 6 million co-transformants of the bait plasmid and a yeast genomic two-hybrid library constructed in the prey plasmid (22). Two positive prey plasmids with inserts of different size contained the complete open reading frame of ARR4, the yeast homologue of the prokaryotic ArsA ATPase (Fig. 1A). In prokaryotes, ArsA is the ATPase subunit of a transporter involved in arsenite efflux (25, 26). In yeast, Arr4p has been characterized as a soluble cytosolic protein involved in heat and metal tolerance (27).

The cytosolic C terminus of most archeal and eukaryotic CLC proteins contains two copies of a conserved cystathione {beta}-synthetase (CBS) domain (28, 29). The occurrence of such CBS domains in a number of functionally and structurally unrelated proteins has remained enigmatic. Because the CT of Gef1p contains two copies of the CBS domain, we assessed the specificity of the interaction with Arr4p and tested the ARR4 prey plasmid against a bait plasmid containing SNF4, a kinase subunit that contains four copies of the CBS domain. We detected no interaction between Arr4p and Snf4p, although Snf4p did interact with its partner subunit Snf1p (Fig. 1A). This pair of interaction partners serves as a well characterized positive control, since the two proteins were used to establish the yeast two-hybrid system (30, 31). We conclude that Arr4p is not generally recruited to tandem CBS domains and interacts specifically with the CT of Gef1p.



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  		FIGURE 1.
Arr4p interacts with the CT of Gef1p. A, growth of co-transformants transformed with empty bait and prey vectors, bait and prey fusions that are known to interact (SNF1/SNF4), the GEF1 CT bait and the ARR4 prey, and a control addressing whether Arr4p binds to the four CBS domains present in Snf4p. Double dropout (D) medium shows serial dilution of transformants, whereas triple dropout (T) medium selects for the interaction between bait and prey. B, co-immunoprecipitation of HA-tagged Gef1p with endogenous or overexpressed Arr4p. 2HA-Gef1p was expressed (from a 2µ plasmid under the control of the PGK1 promoter) in strains expressing or not expressing Arr4p, and detergent-solubilized extracts were immunoprecipitated using an anti-Arr4p antiserum. Immunoprecipitates were probed by Western blotting (WB) using an anti-HA monoclonal antibody. Overexpression of Arr4p was from a CEN plasmid under the control of the MET25 promoter in the absence of methionine. Since Gef1p could not be detected in the input loading control (10 µg of protein), we show anti-HA immunoprecipitates for reference. Relative amounts of loaded material are indicated. C, co-immunoprecipitation of PC-tagged Arr4p (Arr4p-4PC) with HA-tagged CLC protein (2HA-Gef1p), overexpression under the same conditions described for B. Detergent-solubilized extracts were immunoprecipitated using the anti-HA monoclonal antibody, and immunoprecipitates were probed by Western blotting using an anti-PC monoclonal antibody.

 
To investigate the interaction of the Arr4p ATPase with the full-length CLC protein independently from the two-hybrid system we performed co-immunoprecipitation experiments (Fig. 1, B and C). Detergent-solubilized extracts from yeast cells expressing the N-terminally HA-tagged Gef1p channel were immunoprecipitated using an antiserum that we raised against Arr4p. Whereas we were not able to detect the channel in our input control Western blots, the protein was strongly enriched in the immunoprecipitates (Fig. 1B). The amount of Gef1p that we could co-precipitate with the anti-Arr4p serum depended on the amount of Arr4p present in the lysates (none, endogenous levels, overexpression). In the inverse experiment we were able to specifically co-immunoprecipitate a tagged form of Arr4p (Arr4p-4PC) with Gef1p (Fig. 1C). We conclude that Arr4p interacts with Gef1p.

Next, we tried to reconstitute the interaction in vitro employing recombinant proteins. Arr4p N-terminally fused to a decahistidine tag was purified from E. coli by nickel chromatography. The CT of Gef1p was fused to two IgG binding Z domains of protein A and immobilized directly from bacterial lysates (Fig. 2A). We were unable to detect binding of recombinant Arr4p to the CT of Gef1p (Fig. 2B, lane 2). Interestingly, binding was observed in the presence of yeast cytosol (lane 4) but not bacterial cytosol (lane 3). Endogenous Arr4p present in the yeast cytosol did not mediate binding since cytosol from an {Delta}arr4 mutant (lane 5) supported the reaction like cytosol from a protease-deficient strain ({Delta}pep4, lane 4) and wild type cytosol (data not shown). We conclude that binding of recombinant Arr4p to the CT of Gef1p requires a factor present in yeast cytosol and set out to identify the factor.

Fractionation of the cytosol by a 4-kDa cut-off filter and subsequent binding experiments demonstrated that the factor required for binding was either a peptide or a small molecule (Fig. 2C). CBS domains have been suggested to bind ATP, AMP, or S-adenosylmethionine (32) but none of these compounds mediated binding (data not shown). Consistently, apyrase treatment of yeast cytosol to hydrolyze ATP did not impair its ability to support binding (Fig. 2D). We found that the activity persisted after boiling of the cytosol but was sensitive to the presence of EDTA in the binding reaction or to pretreatment with a metal chelating resin (data not shown). These observations suggested that metal ions are required to support the binding reaction. Because of its metal-chelating activity, the decahistidine tag was removed from Arr4p by TEV protease cleavage after purification. Next, we replaced the cytosol by a trace mineral mixture at the concentration normally present in yeast minimal media. This mixture was able to substitute for the yeast cytosol and mediated binding of Arr4p to the CT of Gef1p (Fig. 3A, lanes 1–4). To assess the identity of the required metal, we added different chelators to the binding reaction performed in cytosol (lanes 5–8) and observed that bathocuproine sulfonate (BCS), a Cu(I)-specific chelator (33, 34), as well as bathophenantroline sulfonate, a Fe(II) and Cu(II) chelator (35), abolished binding of Arr4p to the CT of Gef1p. Sensitivity of the binding reaction to BCS suggested that Cu(I) is required to support binding. Supplying Cu(I) by adding a mixture of CuSO4 and ascorbate allowed binding in the absence of cytosol (lanes 9 and 10). However, CuSO4 alone, and thus Cu(II), was able to support binding less efficiently (data not shown).

These results and a second line of experiments raised the possibility that Arr4p is a metal-binding protein. In addition to the metal requirement for binding to the CT of Gef1p, we observed abnormal migration behavior of Arr4p when subjected to SDS-PAGE without SH-reducing reagents in the sample buffer (Fig. 3B). Under these conditions, the protein migrates as a doublet that is converted to one band in the presence of {beta}-mercaptoethanol. Dithiothreitol, glutathione at physiological concentrations, or tris-(2-carboxyethyl)-phosphine, a reducing agent that does not contain SH groups itself, has the same effect (data not shown). Whereas a change in running behavior upon the addition of SH-reducing agents might be indicative of intramolecular disulfide bond formation, disulfide-bonded proteins usually show slower mobility than their reduced counterparts. In the case of the co-chaperone dnaJ,5 migration as a doublet of faster mobility than the fully reduced protein is due to metal coordination by dnaJ (36). Although EDTA did not affect the running behavior of Arr4p (data not shown), we investigated the presence of a metal in recombinant Arr4p purified from E. coli by inductively coupled plasma mass spectrometry. The measurement revealed ~1 mol of zinc/mol of protein. We exploited the correlation between the migration behavior of Arr4p in the absence of SH-reducing agents to ask whether the chelators tested in Fig. 3A converted the protein to the presumably metal-free form also observed in the presence of {beta}-mercaptoethanol (Fig. 3, B and C). BCS but not bathophenantroline sulfonate affected the migration behavior of the protein (Fig. 3C) when the protein was incubated with cytosol in the presence of the respective chelator. Thus, the Cu(I)-specific chelator BCS was specifically able to convert Arr4p to the presumably metal-free form. This result might suggest that copper can replace the zinc bound to the recombinant protein (Fig. 7). We were, however, unable to convert Arr4p into a copper-containing protein in vitro (data not shown). In conclusion, yeast cytosol can support the binding of Arr4p purified from E. coli to the CT of Gef1p, because it contains copper ions accessible to the proteins.



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  		FIGURE 2.
Binding of Arr4p to the CT of Gef1p requires a factor from yeast cytosol. A, schematic depiction of the binding assay. Proteins were purified from E. coli. The CT of Gef1p was immobilized on IgG Sepharose by a double Z domain tag (derived from protein A). Secondary structure elements predicted to form the CBS domains of Gef1p are indicated as black arrows ({beta}-sheets) or gray cylinders ({alpha}-helices) B, binding experiments were performed in binding buffer, bacterial, or yeast cytosol from the genetic background indicated. The matrix was washed, and bound protein was eluted using a high salt buffer. Equal amounts of the eluate from each binding reaction were resolved by SDS-PAGE and stained employing Coomassie Blue. Binding in the presence of wild type cytosol (not shown) was identical to the result using cytosol of a protease-deficient strain ({Delta}pep4). C, binding assay was performed in the presence of cytosol ({Delta}pep4 strain) fractions separated by a 4-kDa cut-off filter. D, binding assay was performed in the presence of cytosol treated with apyrase (4 units/100 µl of cytosol, 15 min, 30 °C).

 



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  		FIGURE 3.
Copper supports the binding of Arr4p to the CT of Gef1p. A, binding assay in the presence of a trace mineral mixture containing 8 µM boric acid, 160 nM CuSO4, 600 nM KI, 1.8µM FeSO4, 2.6µM MnSO4, 700 nM NH4 molybdate, and 1.4µM ZnSO4 (lanes 1 and 2). Note that here and in all subsequent figures, we employed an Arr4p protein where the N-terminal decahistidine tag used to purify the protein had been cleaved off by TEV protease cleavage and removed by nickel chromatography. Additional binding reactions were performed in the presence of {Delta}pep4 cytosol (lanes 3 and 4), cytosol and the indicated chelators (lanes 5–8), or 160 nM CuSO4 and 10 µM ascorbate (lanes 9 and 10). All eluates were transferred to nitrocellulose after SDS-PAGE and probed with an anti-Arr4p serum. B, migration behavior of Arr4p in the absence and presence of {beta}-mercaptoethanol. 2 µg of Arr4p was resolved by SDS-PAGE and stained by Coomassie Blue. C, Arr4p migration behavior after incubation in cytosol ({Delta}pep4 strain) with or without the addition of the Cu(I)-specific chelator BCS. Like SH group-reducing agents and unlike other metal chelators, BCS converts the protein into the single band, slower migrating form. 2 µg of Arr4p was incubated as indicated, resolved by SDS-PAGE, and stained by Coomassie Blue. BPS, bathophenantroline sulfonate.

 
Does the copper-dependent binding of Arr4p to the CT of Gef1p depend on copper availability in the yeast cytosol in vivo? To address this question, we repeated the two-hybrid assay in the presence of BCS (Fig. 4A), an approach that was previously used to demonstrate copper-dependent binding of the cytosolic copper chaperone Atx1p to the N terminus of the copper ATPase Ccc2p (37). In contrast to the most likely copper-independent interaction of the kinase subunits serving as control interaction partners, Snf1p and Snf4p, the interaction between Arr4p and the Gef1p CT was no longer detectable in the presence of BCS in the growth medium (Fig. 4A). Since the running behavior of Arr4p on nonreducing SDS-PAGE gels presumably reflects its metal loading status, we used this property to test the copper loading state of Arr4p under conditions of copper repletion, stress, or deficiency (Fig. 4B). Yeast cells were grown under the respective conditions and lysed by alkaline treatment in the absence of reducing agents. Total cellular protein was precipitated, resolved by nonreducing SDS-PAGE, and probed with an antibody raised against Arr4p. The endogenous protein was found to migrate as a smear under copper replete conditions, as one fast migrating band under copper stress, and as a slower migrating single band under copper depletion (Fig. 4B). The position of this slow migrating form was equivalent to the one observed for the recombinant protein in the presence of SH-reducing reagents or BCS (Fig. 3, B and C). We conclude that the migration behavior of Arr4p reflects cytosolic copper availability in vivo and that the slow migrating form of Arr4p observed under copper depletion cannot bind to the CT of Gef1p.



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  		FIGURE 4.
Binding of Arr4p to the Gef1p CT depends on copper in vivo. A, growth of serial dilutions of co-transformants transformed with the indicated yeast two-hybrid plasmids on triple dropout medium without or with 3 mM BCS. B, analysis of the migration behavior of Arr4p under different growth conditions. Total cellular protein was extracted from yeast cells (expressing an epitope-tagged variant of Gef1p, Gef1p-4PC (48), integrated into the genome under the control of the GEF1 promoter) and grown in dropout medium or in dropout medium supplemented with either 0.5 mM CuSO4 (copper stress) or 3 mM BCS (copper depletion). The extract was resolved by nonreducing SDS-PAGE, and endogenous Arr4p was detected by Western blotting (WB) employing an anti-Arr4p serum.

 
The best characterized function of Gef1p is to ensure copper delivery to the luminal phase of the late secretory pathway under metal depletion conditions (6, 13). Loss of this function leads to the accumulation of the Fet3p oxidase in its apo state and, thus, prevents growth in iron-limiting medium (LIM10 or LIM50, depending on the concentration of iron added to the chelating medium (18)). Since Arr4p does not bind to the CT of Gef1p under copper depletion, we tested the ability of an {Delta}arr4 strain to grow in LIM10 liquid medium (Fig. 5A; identical growth differences were observed when we used LIM50 instead of LIM10 medium (data not shown)). Like the {Delta}gef1 strain, the {Delta}arr4 strain showed impaired growth under iron depletion, although the phenotype was reproducibly less pronounced (Fig. 5A, upper left). In contrast to the {Delta}gef1 strain, this growth phenotype was alleviated by the addition of 20 µM CuSO4 to exclude effects due to the copper-chelating rather than the iron-chelating capacity of the low iron medium (Fig. 5A, upper right). These phenotypes suggest that Arr4p is not an obligatory subunit of the Gef1p ion transport protein, since the {Delta}arr4 strain grows better than the {Delta}gef1 strain when iron but not copper is limiting for growth. The {Delta}arr4 strain has been shown to be slightly heat-sensitive at 37 °C and severely growth-impaired at 40 °C (27). Heat and metal sensitivity of the strain appeared to be additive (27). Thus, we tested the growth of the {Delta}arr4 strain in LIM10 and LIM10 medium supplemented with copper at 37 °C, where the cells were unable to grow (Fig. 5A, bottom), showing that the heat sensitivity and the phenotype observed under iron limitation are additive as well.

As described by Shen et al. (27), we found the {Delta}arr4 strain to be copper-sensitive when the metal was added to synthetic complete medium (Fig. 6E). Because of the growth impairment of the {Delta}gef1 strain at neutral pH (6, 10–12) and because Gef1p is likely to function as a chloride/proton antiporter (7, 8), we tested the effect of copper and heat stress in media buffered to different pH values as described (19). All strains tolerated concentrations of 0.5 and 1 mM exogenous copper well when the medium was buffered to pH 2.7 or 3.8 at growth temperatures of either 30 or 37 °C (data not shown). However, when the medium was buffered to pH 7, we observed pronounced differences (Fig. 5B). At 30 °C (Fig. 5B, upper left), growth of the {Delta}arr4 strain at neutral pH was indistinguishable from the wild type strain, whereas loss of Gef1p was associated with a growth defect as described (6, 10–12). At the same time, {Delta}gef1 strains grew better than wild type or the {Delta}arr4 strain when copper stress and neutral pH were combined Fig. 5B, upper right). At 37 °C (Fig. 5B, lower panels), the heat sensitivity of the {Delta}arr4 strain became visible (in contrast to the same media buffered to more acidic pH values; data not shown). Strikingly, the {Delta}gef1 strain exhibited much more robust growth than even the wild type strain when heat stress, neutral pH of the growth medium, and copper stress were combined (Fig. 5B, lower right). In contrast, loss of Arr4p was incompatible with growth under these conditions. These results suggest that Gef1p dominates copper access to the luminal phase of the endomembrane system; in an alkaline environment, copper and iron are the limiting factors for growth of S. cerevisiae (14), and {Delta}gef1 cells are growth-impaired, presumably because not enough copper reaches the luminal phase. The gain-of-growth phenotype of the {Delta}gef1 strain in the presence of toxic concentrations of copper reveals that the luminal phase of the endomembrane system is the main target of copper stress when cells grow in media buffered to neutral pH. Under these conditions, the absence of Arr4p reduced the ability of GEF1 cells to grow when compared with the wild type strain. Lack of Arr4p and Gef1p together restored growth to wild type levels (Fig. 5B, lower right). We conclude that Arr4p counteracts the function of Gef1p when cellular copper levels are high.



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  		FIGURE 5.
Phenotypes of {Delta}arr4 and {Delta}gef1 strains under iron limitation and copper stress. A, growth curves of the different strains grown in iron-limiting medium (LIM10 (18)) at the indicated temperatures in the absence or presence of added copper (20 µM CuSO4). Circles, wild type (WT); triangles, {Delta}arr4; diamonds, {Delta}gef1; squares, {Delta}arr4{Delta}gef1. The solid line shows a logistic growth curve fitted to the experimental data. Data shown are representative of several independent experiments. B, serial dilutions of the different strains grown in dropout medium were spotted on APG medium (19) buffered to pH 7 with or without the addition of 1 mM CuSO4 (plates incubated at either 30 or 37 °C).

 
The prokaryotic counterpart of Arr4p, ArsA, consists of two mutually homologous P-loop ATPase domains that form a pseudodimeric structure, as revealed by the crystallographic analysis (25). Arr4p is only half the size of ArsA and comprises only one ATPase domain. Thus, Arr4p and its human ortholog most likely exist as homodimers, which is supported by the results of cross-linking as well as gel filtration analyses (27, 38). Metal binding motifs of proteins commonly contain cysteines involved in metal coordination. However, we did not detect a canonical metal binding motif in the Arr4p sequence. To address which cysteines of Arr4p are involved in metal binding, we constructed a homology model of Arr4p (Fig. 6A) based on the ArsA structure (25) using the- "FRankenstein's monster" approach (39), a multistep protocol of comparative modeling. The prokaryotic ArsA ATPase contains an antimony (arsenic)-binding site consistent with the role of this protein in bacterial arsenite resistance (25). This metal binding site is not conserved in Arr4p, since the metal-binding cysteine residues of ArsA (Cys172, Cys113, and Cys442) are missing from the corresponding regions. Thus, the two proteins seem to have acquired different metal binding sites as a consequence of convergent evolution.



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  		FIGURE 6.
Cysteines 285 and 288 are implicated in Arr4p dimerization. A, homology model of Arr4p dimer. Cysteine pairs at positions 240/242 and 285/288 are highlighted in the isolated monomer shown to the right. The location of the cysteine pair at 285/288 in the dimer interface is shown in detail (inset). B, homotypic Arr4p interaction assayed by the two-hybrid system. Bait and prey fusions contained the indicated double mutation. C, blue native gel electrophoresis of recombinant wild type and mutant proteins purified from E. coli visualizes the dimeric form of Arr4p (upper band) and indicates that the C285T/C288T double mutant is dimerization-deficient. The monomeric and dimeric species of bovine serum albumin (BSA) were used as a standard. D, two-hybrid analysis of the interaction between Arr4p and the CT of Gef1p performed and labeled as in Fig. 1A. E, serial dilutions of the {Delta}arr4 strain transformed with the indicated plasmids were spotted on dropout medium without or with the addition of 1 mM CuSO4 (plates incubated at 37 °C for the copper sensitivity). WT, wild type.

 
We used the homology model to identify the spatial location of two pairs of cysteines (Cys240 and Cys242; Cys285 and Cys288) that are present in the yeast as well as human Arr4p but not in ArsA. Cys285 and Cys288 from both monomers were found to form a cluster spanning the dimer interface, whereas Cys240 and Cys242 were found remote from any intramolecular contacts (Fig. 6A). We substituted these two pairs of cysteines with threonine to replace the sulfydryl groups with less reactive hydroxyl groups. In contrast to the C240T/C242T double mutant, the C285T/C288T double mutant was no longer able to form homodimers as assessed by the two-hybrid assay (Fig. 6B) employing mutant ARR4 in the bait as well as the prey fusion plasmids. We further tested this result by blue native gel electrophoresis (Fig. 6C), where recombinant Arr4p purified from E. coli migrated in two species, presumably representing the monomer (lower band) and the homodimer (upper band), which was absent for the C285T/C288T and not the C240T/C242T double mutant. The C285T/C288T double mutant (but not the C240T/C242T double mutant) was unable to interact with the CT of Gef1p as shown by the two-hybrid assay (Fig. 6D) and by the in vitro binding assay using the corresponding recombinant protein purified from E. coli (data not shown). Both mutants were tested for their ability to complement the growth defect of the {Delta}arr4 strain in the presence of high concentrations of exogenous copper at 37 °C (27). The C240T/C242T but not the C285T/C288T double mutant was able to support growth under these conditions (Fig. 6E). Steady-state expression levels of either mutant were comparable with wild type Arr4p when the protein was extracted from yeast cells (data not shown). This indicates that degradation of the C285T/C288T double mutant due to misfolding does not explain the nonfunctionality of the protein. Both mutant proteins contained zinc when purified from E. coli as determined by inductively coupled plasma mass spectroscopy (data not shown). Thus, neither pair of cysteines appears to be directly involved in zinc binding. Mutation of the cysteine pair Cys285/Cys288 abolished homodimerization of Arr4p. Since this mutant form was also unable to bind to the CT of Gef1p, it is tempting to speculate that homodimerization might represent a prerequisite for interaction with the CT of the dimeric protein Gef1p.



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  		FIGURE 7.
Scheme summarizing the effect of metals and reducing agents on the conformation of Arr4p and its ability to bind the CT of Gef1p. Arr4p is schematized as a bracket-shaped object. Arr4p purified as a zinc-containing protein from E. coli. A mobility shift in the presence of reducing agents suggests that cysteines contribute to the metal-binding conformation or the metal-binding site. The identity of these cysteines is unknown. Neither the cysteine pair at residues 240/242 or at residues 285/288 participated directly in the zinc binding site. EDTA did not induce the same mobility shift as reducing agents, but BCS did, provided that the recombinant protein was incubated in yeast cytosol before adding the chelator. Interaction with copper and dimerization were a prerequisite for binding of Arr4p to the CT of Gef1p. Dimerization required the cysteine pair at 285/288. As an alternative hypothesis to the exchange of zinc by copper at the same binding site of Arr4p, a copper-binding site formed between the CT of Gef1p and Arr4p is conceivable.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We identified Arr4p as a new interaction partner of Gef1p in a yeast two-hybrid screen employing the CT of the chloride-transport protein. Co-immunoprecipitation verified the interaction of the ATPase with the full-length membrane protein. CLC chloride channels are a large family of chloride transport proteins involved in chloride movement across the plasma membrane and across intracellular membranes (40). A broad spectrum of physiological functions is associated with CLC channels; they are involved in the stabilization of the membrane potential in muscle cells (CLC-1 (41)), in transepithelial transport in kidney epithelia (CLC-Ka/Kb (42)), and in endosomal or lysosomal acidification (CLC-5/7 (43, 44)). Whereas it seems likely that this diversity in function and subcellular localization is in part mediated by protein-protein interactions with regulatory proteins, virtually nothing is known about such interaction partners. The interaction between Arr4p and Gef1p may be relevant to one branch of intracellular mammalian CLC proteins (e.g. ClC-3, -4, -5) to which Gef1p is most closely related. One of these mammalian homologues has been implicated in the copper loading of ceruloplasmin in hepatocytes (16).

In vitro binding experiments employing recombinantly expressed proteins purified from E. coli revealed that the interaction between the CT of Gef1p and Arr4p required the availability of metal ions (Figs. 2, 3, 4, and 7). The migration behavior of recombinant Arr4p purified from E. coli on nonreducing SDS-polyacrylamide gels suggested that Arr4p may be a metal-binding protein. Indeed, we found that Arr4p purified from E. coli contained zinc. However, this form of the protein was incapable of binding to the CT of Gef1p. The addition of copper to the binding reaction was sufficient to allow binding. Use of the exquisitely specific chelator BCS (33, 34) suggests that copper is not only sufficient but necessary for the binding of Arr4p to the CT of Gef1p in vitro and in vivo. Abnormal metal loading has been observed for other metal binding proteins in heterologous expression systems. For example, copper had to be added to the growth medium of E. coli in order to isolate the recombinant N-terminal copper-binding domains of the Menkes and Wilson disease copper ATPases in a copper-bound state (45). The simplest interpretation of our results is to conclude that the requirement for yeast cytosol in the binding assays reflects the exchange of zinc by copper ions (Fig. 7). Less specific chelators like EDTA and bathophenantroline sulfonate can scavenge the copper ions before the exchange has taken place, but only BCS can remove Cu(I) once coordinated by Arr4p. Similar observations have been described for the copper loading of superoxide dismutase (46). It is, however, conceivable that Arr4p and Gef1p form a common copper-binding site with contributions from both proteins. The copper chaperone Atx1p and the copper ATPase Ccc2p are known to exchange copper via a common interface (47). Since we have so far been unable to purify a copper-bound form of Arr4p, more experiments are required to decide between alternative hypotheses and the model depicted in Fig. 7. We propose a specific role for Arr4p in copper metabolism based on the finding that the addition of either exogenous copper (and not zinc or iron; data not shown) or a copper chelator of unqualified specificity (33), BCS, switches cellular Arr4p between two different conformations that can be distinguished by nonreducing SDS-PAGE (Fig. 4B).

The precise role of the copper-dependent binding of Arr4p to the CT of Gef1p in the localization or function of Gef1p is currently unclear. The copper dependence of the interaction corroborates, however, a role for this chloride transport protein in copper metabolism. Our data place Gef1p and Arr4p in the same biological context. Growth assays with {Delta}arr4 or {Delta}gef1 single and double deletion strains under iron and copper depletion show that both proteins are required for normal growth under iron-limiting conditions (Fig. 5A). For the {Delta}gef1 strain, this defect has been explained by two, possibly additive, effects of Gef1p on the copper loading of Fet3p; the transport activity of Gef1p is thought to affect the driving force for copper uptake to the luminal phase of the endomembrane system (6), and luminal chloride ions directly act as allosteric effectors in the copper loading of Fet3p (13). Our finding that the addition of copper alleviates the growth defect of the {Delta}arr4 strain in low iron medium suggests that, similarly to Gef1p, it is the function of Arr4p in copper metabolism that indirectly causes the sensitivity to iron limitation upon deletion of the corresponding gene. We found that copper influx to the luminal phase of the endomembrane system is a crucial parameter when yeast cells grow in media buffered to neutral pH. Not only is copper influx to this compartment limiting to growth under these conditions (as described previously (6, 14)), but it is also the main cause of copper stress (Fig. 5B, right). Under these conditions, Arr4p and Gef1p seem to functionally counteract each other; loss of Gef1p endowed the cells with the ability to grow better than the corresponding wild type yeast, whereas loss of Arr4p is associated with a copper-sensitive phenotype. Loss of both corresponding genes at the same time results in growth comparable with wild type.

We speculate that Arr4p acts as a copper sensor that associates with membrane proteins, one of them being Gef1p. Our data are consistent with a negative regulation of Gef1p by Arr4p. This could be due to an effect of Arr4p binding on Gef1p-mediated ion transport, on the localization, or on the stability of Gef1p when cellular copper levels are high. Under copper limitation, Arr4p does not bind to the CT of Gef1p and may thus influence copper uptake to the luminal phase of the secretory pathway by binding to another membrane protein. This hypothesis could explain the requirement for Arr4p under iron limitation (Fig. 5A). Thus, Arr4p would be involved in coordinating cytosolic copper levels with copper transport into the luminal phase of the endomembrane system. The high degree of evolutionary conservation between Arr4p and its mammalian counterpart suggests that the protein is also involved in mammalian copper metabolism.


   FOOTNOTES
 
* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB638. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains supplemental information on homology modeling of Arr4p. Back

1 These authors contributed equally to this work. Back

2 An EMBO Young Investigator. Back

3 To whom correspondence should be addressed: Tel.: 49-6221-54-6898; Fax: 49-6221-54-5894; E-mail: b.schwappach{at}zmbh.uni-heidelberg.de.

4 The abbreviations used are: PC, protein C; HA, hemagglutinin; CT, C terminus; BCS, bathocuproine sulfonate. Back

5 M. P. Mayer, personal communication. Back


   ACKNOWLEDGMENTS
 
We thank Dirk Görlich, Matthias Mayer, and Matthias Seedorf for important discussions. We are indebted to Markus Bohnsack, Matthias Seedorf, and members of the Schwappach laboratory for valuable comments on the manuscript.



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