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Arginine Residue at Position 573 in Enterococcus hirae Vacuolar-type ATPase NtpI Subunit Plays a Crucial Role in Na+ Translocation

2002; Elsevier BV; Volume: 277; Issue: 27 Linguagem: Inglês

10.1074/jbc.m200973200

1083-351X

Miyuki Kawano‐Kawada, Kazuei Igarashi, Ichiro Yamato, Yoshimi Kakinuma,

Microbial Fuel Cells and Bioremediation

The 76-kDa NtpI subunit constitutes the membrane-embedded V0 moiety of Enterococcus hirae vacuolar type Na+-ATPase with a 16-kDa NtpK hexamer containing Na+ binding sites. In this study, we investigated the role of an arginine residue, which is highly conserved among the corresponding subunits of bacterial vacuolar-type ATPases, at position 573 of NtpI. Substitution of Glu, Leu, or Gln for Arg-573 abolished sodium transport and sodium-stimulated ATP hydrolysis of the enzyme. The conservative replacement of Arg by Lys lowered both activities about one-fifth of those of the wild type enzyme. We have reported previously on ATP-dependent negative cooperativity for Na+ coupling of this enzyme (Murata, T., Kakinuma, Y., and Yamato, I. (2001) J. Biol. Chem. 276, 48337–48340). The negative cooperativity for the Na+dependence of ATPase activity was weakened by the mutation R573K; the Hill coefficients for the wild type and mutant enzymes at a saturated ATP concentration were 0.22 ± 0.03 and 0.40 ± 0.05, respectively. The Hill coefficients of both enzymes at limited ATP concentrations approached 1. These results indicate that NtpI Arg-573 is indispensable for sodium translocation and for the cooperative features of E. hirae vacuolar-type ATPase. The 76-kDa NtpI subunit constitutes the membrane-embedded V0 moiety of Enterococcus hirae vacuolar type Na+-ATPase with a 16-kDa NtpK hexamer containing Na+ binding sites. In this study, we investigated the role of an arginine residue, which is highly conserved among the corresponding subunits of bacterial vacuolar-type ATPases, at position 573 of NtpI. Substitution of Glu, Leu, or Gln for Arg-573 abolished sodium transport and sodium-stimulated ATP hydrolysis of the enzyme. The conservative replacement of Arg by Lys lowered both activities about one-fifth of those of the wild type enzyme. We have reported previously on ATP-dependent negative cooperativity for Na+ coupling of this enzyme (Murata, T., Kakinuma, Y., and Yamato, I. (2001) J. Biol. Chem. 276, 48337–48340). The negative cooperativity for the Na+dependence of ATPase activity was weakened by the mutation R573K; the Hill coefficients for the wild type and mutant enzymes at a saturated ATP concentration were 0.22 ± 0.03 and 0.40 ± 0.05, respectively. The Hill coefficients of both enzymes at limited ATP concentrations approached 1. These results indicate that NtpI Arg-573 is indispensable for sodium translocation and for the cooperative features of E. hirae vacuolar-type ATPase. vacuolar-type ATPase F0F1-ATPase carbonyl cyanide p-chlorophenylhydrazone 2-([N-cyclohexylamino]ethanesulfonic acid) N,N′-dicyclohexylcarbodiimide 6-amidino-2-naphthyl-4-guanidinobenzoate Hill coefficient nickel-nitrilotriacetic acid The vacuolar H+-ATPase (V-ATPase)1 functions as a proton pump in acidic organelles and plasma membranes of eukaryotic cells as well as in bacteria (1Nelson N. Taiz L. Trends Biochem. Sci. 1989; 14: 113-116Abstract Full Text PDF PubMed Scopus (244) Google Scholar, 2Nelson N. Biochim. Biophys. Acta. 1992; 1100: 109-124Crossref PubMed Scopus (157) Google Scholar, 3Stevens T.H. Forgac M. Annu. Rev. Cell Dev. Biol. 1997; 13: 779-808Crossref PubMed Scopus (523) Google Scholar). We have identified a variant of V-ATPase in Enterococcus hirae, which characteristically transports Na+ or Li+ (4Kakinuma Y. Igarashi K. FEBS Lett. 1990; 271: 97-101Crossref PubMed Scopus (40) Google Scholar). V-ATPase is composed of two structural domains: the hydrophilic/catalytic V1portion and membrane-embedded/ion-translocating V0 portion. In E. hirae V-ATPase, the V1 portion consists of seven subunits (Ntp-A, -B, -C, -D, -E, -F, -G) and V0 two subunits (NtpI and NtpK proteolipid) of molecular masses 76 and 16 kDa, respectively (5Kakinuma Y. Kakinuma S. Takase K. Konishi K. Igarashi K. Yamato I. Biochem. Biophys. Res. Commun. 1995; 195: 1063-1069Crossref Scopus (26) Google Scholar, 6Takase K. Kakinuma S. Yamato I. Konishi K. Igarashi K. Kakinuma Y. J. Biol. Chem. 1994; 269: 11037-11044Abstract Full Text PDF PubMed Google Scholar). V-ATPase thus structurally resembles F-ATPase, which functions as an ATP synthase in mitochondria, chloroplasts, and oxidative bacteria (7Senior A.E. Annu. Rev. Biophys. Chem. 1990; 19: 7-41Crossref PubMed Scopus (329) Google Scholar), and it is widely accepted that the molecular mechanisms of these two ATPases are fundamentally similar (2Nelson N. Biochim. Biophys. Acta. 1992; 1100: 109-124Crossref PubMed Scopus (157) Google Scholar, 8Dimroth P. Wang H. Grabe M. Oster G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4924-4929Crossref PubMed Scopus (135) Google Scholar). In the “rotation catalysis” mechanism of F-ATPase (9Boyer P.D. Annu. Rev. Biochem. 1997; 66: 717-749Crossref PubMed Scopus (1595) Google Scholar), the energy of ATP hydrolysis is converted into the physical force of rotation of the γ subunit, with three ATP being hydrolyzed per rotation (10Noji H. Yasuda R. Yoshida M. Kinosita K., Jr. Nature. 1997; 386: 299-302Crossref PubMed Scopus (1966) Google Scholar). In the F0 portion, it is believed that a rotor ring consisting of c subunits rotates with the γ subunit relative to subunit a, and this rotation couples to proton translocation during ATP synthesis/hydrolysis by F-ATPase (8Dimroth P. Wang H. Grabe M. Oster G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4924-4929Crossref PubMed Scopus (135) Google Scholar, 11Fillingame R.H. Jiang W. Dmitriev O.Y. J. Exp. Biol. 2000; 203: 9-17Crossref PubMed Google Scholar, 12Hutcheon M.L. Duncan T.M. Ngai H. Cross R.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8519-8524Crossref PubMed Scopus (64) Google Scholar, 13Vik S.B. Antonio B.J. J. Biol. Chem. 1994; 269: 30364-30369Abstract Full Text PDF PubMed Google Scholar, 14Kaim G. Matthey U. Dimroth P. EMBO J. 1998; 17: 688-695Crossref PubMed Scopus (47) Google Scholar). In Escherichia coli F-ATPase, there is an arginine residue (Arg-210) that plays a key role in ATP-coupled proton translocation in the fourth transmembrane helix of subunit a. This arginine residue is thought to interact with Asp-61, the proton binding site, in helix 2 of subunit c (8Dimroth P. Wang H. Grabe M. Oster G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4924-4929Crossref PubMed Scopus (135) Google Scholar, 11Fillingame R.H. Jiang W. Dmitriev O.Y. J. Exp. Biol. 2000; 203: 9-17Crossref PubMed Google Scholar, 13Vik S.B. Antonio B.J. J. Biol. Chem. 1994; 269: 30364-30369Abstract Full Text PDF PubMed Google Scholar). In the E. hirae V0portion, ATP-coupled sodium translocation must be catalyzed by the NtpI subunit, which is considered to play the same role as subunit a in F-ATPase, together with six NtpK proteolipids, each having one Na+ binding site (15Murata T. Igarashi K. Kakinuma Y. Yamato I. J. Biol. Chem. 2000; 275: 13415-13419Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Although E. hirae NtpI shows no obvious homology to F-ATPase subunit a, there is an arginine residue (Arg-573) that is conserved among the corresponding subunits of bacterial V-ATPases, in the putative sixth transmembrane segment of NtpI. We expect Arg-573 to interact with Glu-139, the Na+binding site in the fourth transmembrane segment of NtpK (16Takase K. Yamato I. Igarashi K. Kakinuma Y. Biosci. Biotechnol. Biochem. 1999; 63: 1125-1129Crossref PubMed Scopus (7) Google Scholar), in sodium translocation. In this study, we report the effect of Arg-573 mutation on Na+-ATPase activity and found the contribution of NtpI Arg-573 to sodium translocation by E. hirae V-ATPase. The E. hiraestrains used were ATCC 9790 (wild type), obtained from the American Type Culture Collection, Nak1 (nonsense mutant of the ntpAgene) (17Kakinuma Y. Igarashi K. J. Bacteriol. 1990; 172: 1732-1735Crossref PubMed Google Scholar), and the ntpI-deleted strain NID (ntpI::CAT) (18Takase, K., Murata, T., Yamato, I., Igarashi, K., and Kakinuma, Y. (1998) Abstracts of the 71st Annual Meeting of the Japan Biochemical Society Nagoya, Japan, October 14–17, 1998, p. 1099Google Scholar). Cells were grown at 37 °C in a standard complex medium, NaTY (1% Bacto tryptone, 0.5% Bacto yeast extract, 1% glucose, and 0.85% Na2HPO4) (19Kakinuma Y. Harold F.M. J. Biol. Chem. 1985; 260: 2086-2091Abstract Full Text PDF PubMed Google Scholar). When necessary, Na2CO3 was added to this medium to adjust its pH to the appropriate alkaline pH. Replacement of Arg-573 by other amino acids was performed on the wild type ntpI gene in the shuttle vector pHEex (16Takase K. Yamato I. Igarashi K. Kakinuma Y. Biosci. Biotechnol. Biochem. 1999; 63: 1125-1129Crossref PubMed Scopus (7) Google Scholar), containing the erythromycin resistance gene as marker (pHEexI), using a QuikChange site-directed mutagenesis kit (Stratagene). The oligonucleotide primers used were the following, with substitutions underlined: R573E, 5′-CAGCTATACAGAACTAATGGCACTG-3′; R573K, 5′-GTCAGCTATACAAAACTAATGGCACTGGGAATCTCTGG-3′; R573L, 5′-CAGCTATACATTACTAATGGCACTG-3′; and R573Q, 5′-GTCAGCTATACACAACTAATGGCACTGGGAATCTCTGG-3′. Mutations were confirmed by DNA sequencing, using Seq4 × 4 personal sequencing system (Amersham Biosciences), and the mutated fragments were then subcloned back into the vector pHEex. No other mutation was detected in the final products. The plasmids were introduced into strain NID, and the transformants were selected in medium containing 5 μg/ml chloramphenicol and 10 μg/ml erythromycin.