Energetics and Topology of CzcA, a Cation/Proton Antiporter of the Resistance-Nodulation-Cell Division Protein Family
1999; Elsevier BV; Volume: 274; Issue: 37 Linguagem: Inglês
10.1074/jbc.274.37.26065
ISSN1083-351X
AutoresM. Goldberg, T Pribýl, Susanne Juhnke, Dietrich H. Nies,
Tópico(s)Antibiotic Resistance in Bacteria
ResumoThe membrane-bound CzcA protein, a member of the resistance-nodulation-cell division (RND) permease superfamily, is part of the CzcCB2A complex that mediates heavy metal resistance in Ralstonia sp. CH34 by an active cation efflux mechanism driven by cation/proton antiport. CzcA was purified to homogeneity after expression in Escherichia coli, reconstituted into proteoliposomes, and the kinetics of heavy metal transport by CzcA was determined. CzcA is composed of 12 transmembrane α-helices and two large periplasmic domains. Two conserved aspartate and a glutamate residue in one of these transmembrane spans are essential for heavy metal resistance and proton/cation antiport but not for facilitated diffusion of cations. Generalization of the resulting model for the function of CzcA as a two-channel pump might help to explain the functions of other RND proteins in bacteria and eukaryotes. The membrane-bound CzcA protein, a member of the resistance-nodulation-cell division (RND) permease superfamily, is part of the CzcCB2A complex that mediates heavy metal resistance in Ralstonia sp. CH34 by an active cation efflux mechanism driven by cation/proton antiport. CzcA was purified to homogeneity after expression in Escherichia coli, reconstituted into proteoliposomes, and the kinetics of heavy metal transport by CzcA was determined. CzcA is composed of 12 transmembrane α-helices and two large periplasmic domains. Two conserved aspartate and a glutamate residue in one of these transmembrane spans are essential for heavy metal resistance and proton/cation antiport but not for facilitated diffusion of cations. Generalization of the resulting model for the function of CzcA as a two-channel pump might help to explain the functions of other RND proteins in bacteria and eukaryotes. resistance-nodulation-cell division polymerase chain reaction transmembrane helix carbonyl cyanide p-trifluoromethoxyphenyl hydrozone Multiple drug resistant bacteria poses a threat to man's fight against infectious diseases. Some multiple drug resistance systems may detoxify their substrates by transport across the complete cell wall of Gram-negative bacteria, across cytoplasmic membrane, periplasm, and outer membrane. These assumed transenvelope transporters are composed of a pump protein that energizes the transport, in addition to a membrane fusion and an outer membrane-associated protein (1Nikaido H. J. Bacteriol. 1996; 178: 5853-5859Crossref PubMed Scopus (873) Google Scholar, 2Paulsen I.T. Brown M.H. Skurray R.D. Microbiol. Rev. 1996; 60: 575-608Crossref PubMed Google Scholar). The pump protein may be an ATP-binding cassette transporter (3Fath M.J. Kolter R. Microbiol. Rev. 1993; 57: 995-1017Crossref PubMed Google Scholar, 4Saier M.H.J. Microbiol. Rev. 1994; 58: 71-93Crossref PubMed Google Scholar), a transporter of the major facilitator superfamily (5Paulsen I.T. Park J.H. Choi P.S. Saier M.H.J. FEMS Microbiol. Lett. 1997; 156: 1-8Crossref PubMed Scopus (227) Google Scholar), or a resistance-nodulation-cell division (RND)1 protein (4Saier M.H.J. Microbiol. Rev. 1994; 58: 71-93Crossref PubMed Google Scholar, 6Saier M.H. Tam R. Reizer A. Reizer J. Mol. Microbiol. 1994; 11: 841-847Crossref PubMed Scopus (275) Google Scholar, 7Tseng T.-T. Gratwick K.S. Kollman J. Park D. Nies D.H. Goffeau A. Saier M.H.J. J. Mol. Microbiol. Biotechnol. 1999; 1 (, 258–268): 22Google Scholar). The archetype of the RND permease superfamily family is CzcA from the Gram-negative bacterium Ralstonia sp. CH34 (formerly Alcaligenes eutrophus strain CH34) (8Nies D. Mergeay M. Friedrich B. Schlegel H.G. J. Bacteriol. 1987; 169: 4865-4868Crossref PubMed Google Scholar, 9Nies D.H. Nies A. Chu L. Silver S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7351-7355Crossref PubMed Scopus (211) Google Scholar, 10Nies D.H. J. Bacteriol. 1995; 177: 2707-2712Crossref PubMed Google Scholar, 11Rensing C. Pribyl T. Nies D.H. J. Bacteriol. 1997; 179: 6871-6879Crossref PubMed Google Scholar, 12Brim H. Heyndrickx M. De Vos P. Wilmotte A. Springael D. Schlegel H.G. Mergeay M. Syst. Appl. Microbiol. 1999; 22: 258-268Crossref PubMed Scopus (71) Google Scholar). This bacterium contains at least seven heavy metal resistance determinants, located either on the bacterial chromosome or on one of the two indigenous plasmids pMOL28 (163 kilobase pairs) and pMOL30 (238 kb) (8Nies D. Mergeay M. Friedrich B. Schlegel H.G. J. Bacteriol. 1987; 169: 4865-4868Crossref PubMed Google Scholar, 13Taghavi S. Mergeay M. Van der Lelie D. Plasmid. 1997; 37: 22-34Crossref PubMed Scopus (40) Google Scholar, 14Mergeay M. Nies D. Schlegel H.G. Gerits J. Charles P. van Gijsegem F. J. Bacteriol. 1985; 162: 328-334Crossref PubMed Google Scholar, 15Dressler C. Kües U. Nies D.H. Friedrich B. Appl. Environ. Microbiol. 1991; 57: 3079-3085Crossref PubMed Google Scholar, 16Nies A. Nies D.H. Silver S. J. Biol. Chem. 1990; 265: 5648-5653Abstract Full Text PDF PubMed Google Scholar). One of them, the czc-determinant of plasmid pMOL30, mediates inducible resistance to millimolar concentrations of Co2+, Zn2+, and Cd2+ in strain CH34 (8Nies D. Mergeay M. Friedrich B. Schlegel H.G. J. Bacteriol. 1987; 169: 4865-4868Crossref PubMed Google Scholar, 17Nies D.H. Silver S. J. Bacteriol. 1989; 171: 896-900Crossref PubMed Google Scholar). The products of the genes czcA, czcB, and czcC form a membrane-bound protein complex catalyzing an energy-dependent efflux of these three metal cations (9Nies D.H. Nies A. Chu L. Silver S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7351-7355Crossref PubMed Scopus (211) Google Scholar,11Rensing C. Pribyl T. Nies D.H. J. Bacteriol. 1997; 179: 6871-6879Crossref PubMed Google Scholar), probably across the complete envelope. The mechanism of action of CzcCB2A is that of a proton/cation antiporter, and the Km values of the efflux system for the substrate heavy metal cations are also in the millimolar range (10Nies D.H. J. Bacteriol. 1995; 177: 2707-2712Crossref PubMed Google Scholar). Although indirect evidence led to the assumption that CzcA is the central cation/proton antiporter of the CzcCB2A complex (10Nies D.H. J. Bacteriol. 1995; 177: 2707-2712Crossref PubMed Google Scholar), this has not been shown directly. This paper demonstrates that CzcA is a cation/proton antiporter, and develops the model of CzcA as a two-channel pump based on topology studies and the function of CzcA mutant proteins. This model sheds some light on other RND proteins involved in multiple drug resistance of bacteria or with previously unknown functions in mammals. 2T.-T. Tseng, K. S. Gratwick, D. H. Nies, A. Goffeau, and M. H. J. Saier, submitted for publication. Ralstonia sp. strain AE104 (14Mergeay M. Nies D. Schlegel H.G. Gerits J. Charles P. van Gijsegem F. J. Bacteriol. 1985; 162: 328-334Crossref PubMed Google Scholar) is a metal-sensitive, plasmid-free derivative of strain CH34.Escherichia coli K38(pGP1–2) (19Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1074-1078Crossref PubMed Scopus (2459) Google Scholar) was used for expression of czcCBAD derivatives under control of the phage T7 promoter as described (20Nies A. Nies D.H. Silver S. J. Bacteriol. 1989; 171: 5065-5070Crossref PubMed Google Scholar). Tris-buffered mineral salts medium (14Mergeay M. Nies D. Schlegel H.G. Gerits J. Charles P. van Gijsegem F. J. Bacteriol. 1985; 162: 328-334Crossref PubMed Google Scholar) containing 2 g/liter sodium gluconate was used for testing metal resistance and growth of Ralstonia. E. coli was cultivated in Luria broth (21Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd. Ed. Cold Spring Harbor Laboratory., Cold Spring Harbor, NY1989Google Scholar). Analytical grade salts of CdCl2·H2O, ZnCl2, and CoCl2·6 H2O were used to prepare 1m stock solutions, which were sterilized by filtration. Solid Tris-buffered medium contained 2 g/liter agar. Minimal inhibitory concentrations were determined as described (14Mergeay M. Nies D. Schlegel H.G. Gerits J. Charles P. van Gijsegem F. J. Bacteriol. 1985; 162: 328-334Crossref PubMed Google Scholar) using Tris-buffered mineral salts medium. Protein concentrations were determined using the Bradford method (22Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar), unless otherwise stated. Standard molecular genetic techniques were used (8Nies D. Mergeay M. Friedrich B. Schlegel H.G. J. Bacteriol. 1987; 169: 4865-4868Crossref PubMed Google Scholar, 21Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd. Ed. Cold Spring Harbor Laboratory., Cold Spring Harbor, NY1989Google Scholar). Transformation of E. coli strains was conducted as described previously (8Nies D. Mergeay M. Friedrich B. Schlegel H.G. J. Bacteriol. 1987; 169: 4865-4868Crossref PubMed Google Scholar). For expression of czcCBAD derivatives under control of the lac promoter in Ralstonia strain AE104, the plasmid pT7–5-derivatives containing the various czc constructs were cut with Eco RI and Xba I and cloned into the broad host range plasmid pVDZ′2 (23Deretic V. Chandrasekharappa S. Gill J.F. Chatterjee D.K. Chakrabarty A. Gene. 1987; 57: 61-72Crossref PubMed Scopus (58) Google Scholar). The pVDZ′2-derivatives were transformed into E. coli S17–1 (24Simon R. Priefer U. Pühler A. Bio/Technology. 1983; 1: 784-791Crossref Scopus (5658) Google Scholar) and transferred into Ralstonia AE104 by conjugation as described (8Nies D. Mergeay M. Friedrich B. Schlegel H.G. J. Bacteriol. 1987; 169: 4865-4868Crossref PubMed Google Scholar). For reporter gene fusions, fusion vector pECD500 (11Rensing C. Pribyl T. Nies D.H. J. Bacteriol. 1997; 179: 6871-6879Crossref PubMed Google Scholar) and E. coli CC118 were used (25Manoil C. J. Bacteriol. 1990; 172: 1035-1042Crossref PubMed Google Scholar). All fusions were done immediately downstream of an arginine or lysine residue of CzcA. Activity of alkaline phosphatase (25Manoil C. J. Bacteriol. 1990; 172: 1035-1042Crossref PubMed Google Scholar) was determined in triplicate as published previously. Mutations in the czcA gene were constructed using PCR by an overlap extension method (26Mikaelian I. Sergeant A. Nucleic Acids Res. 1992; 20: 376Crossref PubMed Scopus (211) Google Scholar) as published previously (11Rensing C. Pribyl T. Nies D.H. J. Bacteriol. 1997; 179: 6871-6879Crossref PubMed Google Scholar). The 5′ part of the internal 1,034-base pair Nhe I-Mun I internal fragment of czcA (position 3,489 to 4,523) (9Nies D.H. Nies A. Chu L. Silver S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7351-7355Crossref PubMed Scopus (211) Google Scholar) was amplified from plasmid pECD110 (9Nies D.H. Nies A. Chu L. Silver S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7351-7355Crossref PubMed Scopus (211) Google Scholar) using a primer corresponding to the sequence at the Nhe I site (TAGAGGATCCCGAACGGCTAGCGTCGTA, Nhe I primer) and a primer corresponding to the mutated region. These were (mutations underlined) TCGAAAACAGTGTGAGGCGA for C417S, TGGCGCGTGCGCAGGAACA for H423R, AGGAACGCCATGGCCGGC for H427R, AGGAACACCGTGGCCGGC for H428R, TCCGAGCGGTTCCGTGAGGT for H439R, GTGGTGATTGTCGACAACTGTGTG for E415D, GTGGTGATTGTCCAAAACTGTGTG for E415Q, GGCGCGCTCAACTTCGGCATC for D402N, and ATCATCATCAATGGCGCGGTG for D408N. The 3′-part of the 1,034 contained the region from the mutation to the Mun I site. These fragments were amplified from pECD110 (9Nies D.H. Nies A. Chu L. Silver S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7351-7355Crossref PubMed Scopus (211) Google Scholar) with primers inverse to the primers listed above and a Mun I region primer (AAAGGATCCCACGAACAATTGACACC, Mun I-primer). After purification (Wizard PCR preps, Promega, Madison, WI), each pair of PCR fragments was used as a template in another PCR reaction with the Nhe I and Mun I primers, which both contained additional Bam HI sites at their ends to facilitate cloning. The resulting DNA fragment was purified, digested with Bam HI, and cloned into pUC19 (27Yanisch-Perron C. Vieira J. Messing J. Gene. 1985; 33: 103-119Crossref PubMed Scopus (11472) Google Scholar). All final PCR products were sequenced to verify the exchange and to check for PCR-mediated unwanted changes. To facilitate the exchange of the wild type Nhe I-Mun I fragment against the mutated fragments, the respective fragment from plasmid pECD110 was first deleted and a kanamycin resistance gene was inserted instead leading to plasmid pECD451. This was done as published (11Rensing C. Pribyl T. Nies D.H. J. Bacteriol. 1997; 179: 6871-6879Crossref PubMed Google Scholar). Next, the mutated Nhe I-Mun I fragments were inserted into pECD451 instead of the kanamycin resistance gene. The resulting plasmids were verified by restriction endonuclease digestion using several enzymes, and correct expression of the mutant proteins was demonstrated by T7 expression (19Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1074-1078Crossref PubMed Scopus (2459) Google Scholar). The czcA gene was PCR-amplified as an Eco RI-Bam HI fragment from plasmid pECD110 (9Nies D.H. Nies A. Chu L. Silver S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7351-7355Crossref PubMed Scopus (211) Google Scholar) and cloned into the vector pASK3 (Institut für Bioanalytik, Göttingen, Germany) in E. coli BL21 (Stratagene Europe, Amsterdam, Netherlands). The resulting plasmid pECD559 was verified by DNA sequencing. For each expression, the bacterial strain was freshly transformed. E. coli BL21(pECD559) was cultivated for 16 h at 30 °C and diluted into 1 liter of fresh Luria broth (21Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd. Ed. Cold Spring Harbor Laboratory., Cold Spring Harbor, NY1989Google Scholar). The cultures were cultivated with shaking at 30 °C until the optical density at 600 nm reached 1.0. CzcA production was induced with 100 μg of anhydrotetracyclin/liter of medium. The cells were cultivated for an additional 3 h with shaking at 30 °C and harvested by centrifugation. The pellet was washed and suspended in 10 ml of buffer W (100 mm Tris-HCl buffer (pH 8.0), 1 mm EDTA), treated twice with the French press (SLM Aminco, SOPRA GmbH, Germany; 1,000 psi) in the presence of protease inhibitor (1 mm phenylmethylsulfonyl fluoride) and DNase (10 μg/liter), and cell debris was removed by centrifugation for 15 min at 20,000 × g. The membrane fraction of the resulting supernatant was harvested by ultracentrifugation (1.5 h, 100,000 × g) and suspended in buffer W at 10 g of protein/liter (28Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar, 29Bensadown A. Weinstein D. Anal. Biochem. 1976; 70: 241-250Crossref PubMed Scopus (2740) Google Scholar). CzcA was solubilized from membranes of the E. coli host using 1 g of n-dodecyl-maltoside/g of membrane protein and 3.5 g ofl-α-phosphatidylcholine, β-linoyl-γ-palmitoyl/liter (stirred for 30 min at 23 °C), and membrane debris was removed (30 min, 100,000 × g). CzcA was purified on a strep-tactin-Sepharose column (bed volume 1 ml) equilibrated with buffer W containing 0.1 mm n-dodecyl-maltoside and 0.2 g of phospholipid/liter. After the column was washed with 12 bed volumes buffer W containing 0.1 mm n-dodecyl-maltoside and 0.2 g of phospholipid/liter, CzcA was eluted using buffer W containing 0.1 mm n-dodecyl-maltoside, 0.2 g of phospholipid/liter, and 2.0 mm desthiobiotin. Aliquots from the different steps of the purification procedure were loaded on a SDS gel (30Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). Soybeanl-α-phosphatidylcholine (type II-S, 17% phosphatidylcholine) was suspended in Tris buffer (20 mmTris-HCl, pH 7.0, 2 mm dithiothreitol, β-d-octylglucoside (15 g/liter)) to yield a lipid concentration of 10 g/liter. Subsequently, β-d-octylglucoside was removed by dialysis against 20 mm Tris-HCl, pH 7.0, 2 mm dithiothreitol, and the resulting liposomes were frozen in liquid nitrogen and stored at −80 °C. For reconstitution, the liposomes were thawed and extruded through a 400-nm filter (31Mayer L.D. Hope M.J. Cullis P.R. Biochim. Biophys. Acta. 1986; 858: 161-168Crossref PubMed Scopus (1576) Google Scholar). Triton X-100 (0.45% w/v) was added, and total solubilization of lipids was determined by measuring changes in adsorbance at 540 nm (32Rigaud J.-L. Pitard B. Philippot J.R. Schuber F. Liposomes as Tools in Basic Research and Industy. CRC Press, Inc., Boca Raton, FL1995: 71-88Google Scholar, 33Jung H. Tebbe S. Schmid R. Junk K. Biochemistry. 1998; 37: 11083-11088Crossref PubMed Scopus (79) Google Scholar). Detergent-destabilized liposomes were mixed with purified CzcA in a 100:1 ratio (w/w) and incubated at room temperature under gentle agitation for 15 min. Detergent was removed by adding Bio-Beads SM-2 at a final concentration of 80 mg/ml (34Holloway P.W. Anal. Biochem. 1973; 53: 304-308Crossref PubMed Scopus (641) Google Scholar). After 30 min of incubation, Bio-Beads were removed by filtration on glass silk, and incubation was continued with fresh Bio-Beads for an additional 30 min at 23 °C, and overnight at 4 °C. The turbid proteoliposome suspension was dialyzed two times against 20 mm Tris-HCl, pH 7.0, 2 mm dithiothreitol at 4 °C, and concentrated by centrifugation at 300,000 ×g for 45 min. For NH4Cl loading, the proteoliposomes (pellet) were suspended in 4 ml of 20 mmTris-HCl buffer, pH 7.0 (containing 0.5 mNH4Cl) and incubated on ice for 30 min, followed by a second ultracentrifugation at 300,000 × g for 45 min. The pellet was suspended in 20 mm Tris-HCl buffer, pH 7.0 (containing 0.5 m NH4Cl). Light scattering indicated a mean diameter of the resulting proteoliposomes of about 500 nm. This calculates to a liposome volume of 65.4 attoliter, an outer surface area of 785,000 nm2 and an inner surface area of 736,000 nm2. With a surface area of 0.23 nm2/phospholipid (35Small D. Zoeller R.A. Meyer R.A. Encyclopedia of Molecular Biology and Molecular Medicine. 3. VCH Verlagsgesellschaft mbH, Weinheim, Germany1996: 442-462Google Scholar), each liposome contained 6.61·106 phospholipids/liposome. With 1 g of CzcA (116,611 g/mol)/100 g of phospholipids (734 g/mol), about 417 CzcA proteins should have been present in each proteoliposome. The loaded proteoliposomes were frozen in liquid nitrogen and stored at −80 °C. As published (10Nies D.H. J. Bacteriol. 1995; 177: 2707-2712Crossref PubMed Google Scholar,36Rosen B.P. Methods Enzymol. 1986; 125: 328-336Crossref PubMed Scopus (113) Google Scholar), 1 μl of proteoliposomes were diluted into 2 ml of buffer C (10 mm Tris-HCl (pH 8.0), 0.5 m choline chloride, 5 mm MgCl2) containing 2 μmacridine orange (3,6 bis-dimethylaminoacridine). Acridine orange fluorescence was measured using an excitation wavelength of 490 nm and an emission wavelength of 530 nm (SFM25 spectrofluorometer, Kontron, Zürich, Switzerland) in stirred cuvettes at 24 °C. Cation uptake experiments using the filtration method were performed as described (17Nies D.H. Silver S. J. Bacteriol. 1989; 171: 896-900Crossref PubMed Google Scholar, 36Rosen B.P. Methods Enzymol. 1986; 125: 328-336Crossref PubMed Scopus (113) Google Scholar) with some modifications. The NH4Cl-containing proteoliposomes were diluted into Tris-choline buffer (0.5 m choline chloride, 20 mm Tris (pH 9.0)) to a final volume of 30 μl. After 1 min (5 min in inhibitor experiments), cation uptake was started by the addition of the radioactive cations 65Zn2+,57Co2+, or 109Cd2+(Amersham Pharmacia Biotech, Braunschweig, Germany), and the reaction mixture was incubated at 30 °C. Samples (5 μl) were filtered through membrane filters (pore size 0.45 μm, Schleicher & Schuell) and rinsed with 0.3 ml of Tris-choline buffer containing 10 mm EDTA and 10 mm Mg2+. The radioactivity that remained on the membrane filter was determined with a scintillation counter (LS6500, Beckman, München, Germany). Control liposomes without CzcA were prepared using the same amounts of phospholipids but no CzcA. To calculate a "mol zinc/mol CzcA" value with these negative controls, the amount of zinc accumulated by control liposomes was divided by the CzcA content of the CzcA-containing proteoliposomes in the parallel experiment, which was 0.5 μg/sample. With 417 CzcA molecules/proteoliposome and an internal volume of 65.4 attoliter/proteoliposome, 1 Zn2+/CzcA transported into the proteoliposome corresponds to an internal zinc concentration (ci) of 10 μm. CzcA was purified to homogeneity (Fig.1). The first amino acids of CzcA were determined as MFE as expected from DNA sequence analysis (9Nies D.H. Nies A. Chu L. Silver S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7351-7355Crossref PubMed Scopus (211) Google Scholar). CzcA was reconstituted into proteoliposomes, which were incubated in 100 mm Tris-HCl, pH 5.0. At 1 mm Zn2+, CzcA proteoliposomes accumulated about 70 mol of Zn2+/mol of CzcA (ci = 0.7 mm) more within the first 15 s than control liposomes (Fig.2 A). In the following 2 min, another 100 mol of Zn2+/CzcA (final ci = 1.7 mm) were accumulated, whereas binding of zinc by control liposomes did not increase with time. The rapid transport of zinc by CzcA proteoliposomes could be inhibited by 100 μmof the protonophore FCCP (Fig. 2 A) or using Tris buffer, pH 5.0, instead of pH 7.0 for dilution (data not shown). Thus, this rapid transport was probably driven by the zinc concentration gradient across the proteoliposome membrane. Increased amounts of CzcA proteoliposomes led to increased metal transport, but increased amounts of control liposomes did not (Fig. 2 B). Thus, CzcA was functional, catalyzed a proton-independent fast facilitated diffusion of zinc and a proton-dependent slower transport of zinc into the proteoliposomes.Figure 2Activity of CzcA-containing proteoliposomes. A volume of 3 μl of proteoliposomes (3 g of CzcA/liter) in 100 mm Tris-HCl, pH 5.0, was diluted into 100 mm Tris-HCl buffer, pH 7.0 (panels A and B), or proteoliposomes charged with 0.5 mNH4Cl were diluted into choline buffer, pH 9.0 (panels C and D). After 1 or 5 min,65Zn2+ was added. 5-μl samples (containing 0.5 μg of CzcA each) were filtrated, washed, and used to calculate the mols of zinc transported into the proteoliposomes per mol of CzcA (molecular mass of CzcA-strep-tag, 116,611 Da). Control liposomes did not contain CzcA but were prepared in a parallel fashion, contained the same amount of phospholipids, and were adjusted to a concentration giving the same acridine orange fluorescence quench level as CzcA proteoliposomes. To calculate a "mol of zinc/mol of CzcA" value with these negative controls, the amount of zinc accumulated by control liposomes was divided by the CzcA content of the CzcA-containing proteoliposomes in the parallel experiment, which was 0.5 μg/sample.Panel A, the uptake of CzcA proteoliposomes (•) in the Tris system was compared with CzcA-containing proteoliposomes in the presence of 100 μm FCCP (▪). After 10-fold dilution, 1 mm of zinc was added after 1 min. The control (○) were liposomes without CzcA. Panel B, uptake of zinc (1 mm) by CzcA-containing proteoliposomes diluted 10-fold (•) or 6-fold (▪). Control liposomes were diluted 10- (○), 6- (▪), or 2.5-fold (Δ). Please note that these data are given in "nmol of zinc/sample" to show the differences between the various amounts of proteoliposomes and liposomes. Panel C, CzcA-containing proteoliposomes (•) and control liposomes in the ammonium chloride/choline system (○). Panel D, uptake of 3 (•), 1 (▪), or 0.5 mm of zinc (▴). The values obtained for the liposome control were subtracted from the CzcA proteoliposome values to yield a zero point.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To yield a stronger proton gradient, the proteoliposomes were loaded with 0.5 m NH4Cl; diffusion of NH3out of the liposome leaves protons inside and generates a proton gradient. A stable gradient for at least 20 min was shown with acridine orange fluorescence quenching experiments (data not shown). Ammonium chloride-charged CzcA proteoliposomes accumulated Zn2+rapidly (30-fold, Fig. 2 C), whereas control liposomes did not. CzcA-dependent uptake of zinc into ammonium-charged proteoliposomes could be partially inhibited with 100 μmFCCP or 500 μm carbonylcyanide m-chlorophenylhydrazone, but neither with 10 μm FCCP nor 50 μm carbonylcyanide m-chlorophenylhydrazone (data not shown). The high concentration of uncoupler needed in these experiments might be the result of the very high concentration of membranes used in the experiments (Fig. 2). Velocity of zinc uptake was two-fold higher when 3 mm zinc was used as the substrate instead of 1 mm, however, nearly no uptake was measured with 0.5 mm zinc (Fig. 2 D). The velocity of zinc transport by CzcA followed a sigmoidal substrate saturation curve (Fig. 3 A). A Vmax value of 385 s−1 (TableI) was calculated from the velocities at 10 and 5 mm zinc, and this velocity was used for a Hill plot (Fig. 3 B) yielding a cooperativity constant n = 2 and a K50 of 6.6 mm (Table I). Concentration higher than 10 mmgave no significant uptake, probably due to the toxic effect of zinc on the integrity of the proteoliposomes (data not shown).Table IHeavy metal cation transport by CzcAMetal ionnKm/K50Vmaxmm1/secZn2+1.966.6385Co2+1.9118.5100Cd2+(1)7.728The velocity of metal cation uptake was determined for all three metals. For zinc and cobalt, the substrate saturation curve was sigmoidal. For zinc, the Vmax was determined using the velocities at 5 and 10 mm; for cobalt, it was the velocity at 50 mm. Higher concentrations of either metal inactivated the proteoliposomes. With this Vmaxvalue, a Hill plot using the equation ln(V/Vmax −V) = n · ln S −n · ln K50 was performed yielding the resulting values. For cadmium, concentration higher than 5 mm inactivated the proteoliposomes, and no significant transport could be measured with concentrations lower than 1 mm. Within this range of concentrations, the Lineweaver-Burk plot was linear and yielded the listed Km and Vmax values. Open table in a new tab The velocity of metal cation uptake was determined for all three metals. For zinc and cobalt, the substrate saturation curve was sigmoidal. For zinc, the Vmax was determined using the velocities at 5 and 10 mm; for cobalt, it was the velocity at 50 mm. Higher concentrations of either metal inactivated the proteoliposomes. With this Vmaxvalue, a Hill plot using the equation ln(V/Vmax −V) = n · ln S −n · ln K50 was performed yielding the resulting values. For cadmium, concentration higher than 5 mm inactivated the proteoliposomes, and no significant transport could be measured with concentrations lower than 1 mm. Within this range of concentrations, the Lineweaver-Burk plot was linear and yielded the listed Km and Vmax values. Cobalt transport by CzcA was much slower than zinc transport. To measure any significant transport, 1.5 μl of proteoliposomes instead of 0.3 μl had to be used, and the cobalt concentration had to be raised to 10 mm (Fig.4 A). The substrate saturation of cobalt transport by CzcA was again sigmoidal (Fig. 3 A), and, due to a toxic effect, no uptake was detectable at concentrations higher than 50 mm. Using the velocity measured at 50 mm, a Hill plot was performed (Fig. 3 B) yielding n = 2 and a K50 of 18.5 mm (Table I). Cadmium transport by CzcA was even slower than cobalt uptake (Fig. 4 B). No uptake could be detected at concentrations lower than 1 mm or higher than 5 mm. In this narrow range of substrate concentration (Fig.3 A), a Lineweaver-Burk plot was linear and yielded a Vmax of 28 s−1 and a Km of 7.7 mm (Table I). Computer predictions (data not shown) indicated 12 hydrophobic peaks in the amino acid sequence of CzcA, which might resemble membrane-spanning α-helices (TMHs), and two large hydrophilic regions between TMH I/II and TMH VII/VIII, respectively. The specific activities of CzcA::PhoA translational fusions (Table II) gave evidence for (i) a cytoplasmic location of both termini of CzcA, (ii) a periplasmic location of both large hydrophilic domains, (iii) the presence of TMHs I (between N terminus and large hydrophilic domain 1), II, III, IV, VII, VIII, IX, XI and XII. No fusion could be isolated between TMHs IX and X, and the low specific activity of the fusion between TMHs V and VI (position 475) gave no evidence for a periplasmic location of this region (Table II).Table IISpecific activity and proposed localization of CzcA::PhoA translational fusionsPositionSpecific activity of PhoA fusions, d.w.2-aMean ± S.D. of triplicate determinations are shown.Amino acid residue of CzcAPredicted topology of CzcAunits/mg13N terminus0.15 ± 0.0233Large hydrophilic domain 12.14 ± 0.1064Large hydrophilic domain 12.60 ± 0.03192Large hydrophilic domain 14.31 ± 0.14334Large hydrophilic domain 13.80 ± 0.01345Large hydrophilic domain 13.97 ± 0.07366TMH II/III0.50 ± 0.00390TMH III/IV2.89 ± 0.03419TMH IV2.38 ± 0.17449TMH IV/V0.37 ± 0.01475TMH V/VI0.28 ± 0.03510TMH VI/VII0.35 ± 0.02532TMH VI/VII0.32 ± 0.01557TMH VII0.46 ± 0.02880Large hydrophilic domain 20.99 ± 0.04927TMH VIII/IV0.20 ± 0.02980TMH X/XI0.16 ± 0.041008TMH XI/XII2.13 ± 0.021038C terminus0.16 ± 0.032-a Mean ± S.D. of triplicate determinations are shown. Open table in a new tab The highly conserved (Fig. 5) aminoacyl residues Asp-402, Asp-408, and Glu-415 of CzcA were mutated to D402N, D408N, E415Q, and E415D. The predicted structure of CzcA leaves as possible metal-binding residues 3 histidine residues and 1 cysteine residue between TMH IV/V, these were m
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