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. 1991 Apr;173(8):2639–2643. doi: 10.1128/jb.173.8.2639-2643.1991

Essential residues in the polar loop region of subunit c of Escherichia coli F1F0 ATP synthase defined by random oligonucleotide-primed mutagenesis.

D Fraga 1, R H Fillingame 1
PMCID: PMC207831  PMID: 2013577

Abstract

The conserved, polar loop region of subunit c of the Escherichia coli F1F0 ATP synthase is postulated to function in the coupling of proton translocation through F0 to ATP synthesis in F1. We have used a random mutagenesis procedure to define the essential residues in the region. Oligonucleotide-directed mutagenesis was carried out with a random mixture of mutant oligonucleotides, the oligonucleotide mixture being generated by chemical synthesis by using phosphoramidite nucleotide stocks that were contaminated with the other three nucleotides. Thirty mutant genes coding single-amino-acid substitutions in the region between Glu-37 and Leu-45 of subunit c were tested for function by analyzing the capacity of plasmids carrying the mutant genes to complement a Leu-4----amber subunit c mutant. All substitutions at the conserved Arg-41 residue resulted in loss of oxidative phosphorylation, i.e., transformants could not grow on a succinate carbon source. The other conserved residues were more tolerant to substitution, although most substitutions did result in impaired growth on succinate. We conclude that Arg-41 is essential in the function of the polar loop and that the ensemble of other conserved residues collectively maintain an optimal environment required for that function.

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Selected References

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  1. Cozens A. L., Walker J. E. The organization and sequence of the genes for ATP synthase subunits in the cyanobacterium Synechococcus 6301. Support for an endosymbiotic origin of chloroplasts. J Mol Biol. 1987 Apr 5;194(3):359–383. doi: 10.1016/0022-2836(87)90667-x. [DOI] [PubMed] [Google Scholar]
  2. Deckers-Hebestreit G., Schmid R., Kiltz H. H., Altendorf K. F0 portion of Escherichia coli ATP synthase: orientation of subunit c in the membrane. Biochemistry. 1987 Aug 25;26(17):5486–5492. doi: 10.1021/bi00391a041. [DOI] [PubMed] [Google Scholar]
  3. Derbyshire K. M., Salvo J. J., Grindley N. D. A simple and efficient procedure for saturation mutagenesis using mixed oligodeoxynucleotides. Gene. 1986;46(2-3):145–152. doi: 10.1016/0378-1119(86)90398-7. [DOI] [PubMed] [Google Scholar]
  4. Dewey R. E., Schuster A. M., Levings C. S., Timothy D. H. Nucleotide sequence of F(0)-ATPase proteolipid (subunit 9) gene of maize mitochondria. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1015–1019. doi: 10.1073/pnas.82.4.1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Foster D. L., Fillingame R. H. Stoichiometry of subunits in the H+-ATPase complex of Escherichia coli. J Biol Chem. 1982 Feb 25;257(4):2009–2015. [PubMed] [Google Scholar]
  6. Fraga D., Fillingame R. H. Conserved polar loop region of Escherichia coli subunit c of the F1F0 H+-ATPase. Glutamine 42 is not absolutely essential, but substitutions alter binding and coupling of F1 to F0. J Biol Chem. 1989 Apr 25;264(12):6797–6803. [PubMed] [Google Scholar]
  7. Girvin M. E., Hermolin J., Pottorf R., Fillingame R. H. Organization of the F0 sector of Escherichia coli H+-ATPase: the polar loop region of subunit c extends from the cytoplasmic face of the membrane. Biochemistry. 1989 May 16;28(10):4340–4343. doi: 10.1021/bi00436a032. [DOI] [PubMed] [Google Scholar]
  8. Hensel M., Deckers-Hebestreit G., Schmid R., Altendorf K. Orientation of subunit c of the ATP synthase of Escherichia coli--a study with peptide-specific antibodies. Biochim Biophys Acta. 1990 Mar 15;1016(1):63–70. doi: 10.1016/0005-2728(90)90007-q. [DOI] [PubMed] [Google Scholar]
  9. Hoppe J., Schairer H. U., Friedl P., Sebald W. An Asp-Asn substitution in the proteolipid subunit of the ATP-synthase from Escherichia coli leads to a non-functional proton channel. FEBS Lett. 1982 Aug 16;145(1):21–29. doi: 10.1016/0014-5793(82)81198-8. [DOI] [PubMed] [Google Scholar]
  10. Hoppe J., Schairer H. U., Sebald W. The proteolipid of a mutant ATPase from Escherichia coli defective in H+-conduction contains a glycine instead of the carbodiimide-reactive aspartyl residue. FEBS Lett. 1980 Jan 1;109(1):107–111. doi: 10.1016/0014-5793(80)81321-4. [DOI] [PubMed] [Google Scholar]
  11. Hoppe J., Sebald W. The proton conducting F0-part of bacterial ATP synthases. Biochim Biophys Acta. 1984 Apr 9;768(1):1–27. doi: 10.1016/0304-4173(84)90005-3. [DOI] [PubMed] [Google Scholar]
  12. Kraft R., Tardiff J., Krauter K. S., Leinwand L. A. Using mini-prep plasmid DNA for sequencing double stranded templates with Sequenase. Biotechniques. 1988 Jun;6(6):544-6, 549. [PubMed] [Google Scholar]
  13. Lim W. A., Sauer R. T. Alternative packing arrangements in the hydrophobic core of lambda repressor. Nature. 1989 May 4;339(6219):31–36. doi: 10.1038/339031a0. [DOI] [PubMed] [Google Scholar]
  14. Miller M. J., Fraga D., Paule C. R., Fillingame R. H. Mutations in the conserved proline 43 residue of the uncE protein (subunit c) of Escherichia coli F1F0-ATPase alter the coupling of F1 to F0. J Biol Chem. 1989 Jan 5;264(1):305–311. [PubMed] [Google Scholar]
  15. Miller M. J., Oldenburg M., Fillingame R. H. The essential carboxyl group in subunit c of the F1F0 ATP synthase can be moved and H(+)-translocating function retained. Proc Natl Acad Sci U S A. 1990 Jul;87(13):4900–4904. doi: 10.1073/pnas.87.13.4900. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mosher M. E., White L. K., Hermolin J., Fillingame R. H. H+-ATPase of Escherichia coli. An uncE mutation impairing coupling between F1 and Fo but not Fo-mediated H+ translocation. J Biol Chem. 1985 Apr 25;260(8):4807–4814. [PubMed] [Google Scholar]
  17. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Senior A. E. ATP synthesis by oxidative phosphorylation. Physiol Rev. 1988 Jan;68(1):177–231. doi: 10.1152/physrev.1988.68.1.177. [DOI] [PubMed] [Google Scholar]
  19. Taylor J. W., Ott J., Eckstein F. The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res. 1985 Dec 20;13(24):8765–8785. doi: 10.1093/nar/13.24.8765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Walker J. E., Saraste M., Gay N. J. The unc operon. Nucleotide sequence, regulation and structure of ATP-synthase. Biochim Biophys Acta. 1984 Sep 6;768(2):164–200. doi: 10.1016/0304-4173(84)90003-x. [DOI] [PubMed] [Google Scholar]
  21. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  22. Young E. G., Hanson M. R., Dierks P. M. Sequence and transcription analysis of the Petunia mitochondrial gene for the ATP synthase proteolipid subunit. Nucleic Acids Res. 1986 Oct 24;14(20):7995–8006. doi: 10.1093/nar/14.20.7995. [DOI] [PMC free article] [PubMed] [Google Scholar]

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