Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1991 Nov;173(22):7240–7248. doi: 10.1128/jb.173.22.7240-7248.1991

Targeted mutagenesis of the b subunit of F1F0 ATP synthase in Escherichia coli: Glu-77 through Gln-85.

K A McCormick 1, B D Cain 1
PMCID: PMC209231  PMID: 1682301

Abstract

Subunit b of Escherichia coli F1F0 ATP synthase contains a large hydrophilic region thought to be involved in the interaction between F1 and F0. Oligonucleotide-directed mutagenesis was used to evaluate the functional importance of a segment of this region from Glu-77 through Gln-85. The mutagenesis procedure employed a phagemid DNA template and a doped oligonucleotide primer designed to generate a predetermined collection of missense mutations in the target segment. Sixty-one mutant phagemids were identified and shown to contain nucleotide substitutions encoding 37 novel missense mutations. Mutations were isolated singly or in combinations of up to four mutations per recombinant phagemid. F1F0 ATP synthase function was studied by mutant phagemid complementation of a novel E. coli strain in which the uncF (b) gene was deleted. Complementation was assessed by observing growth on solid succinate minimal medium. Many phagemid-encoded uncF (b) gene mutations in the targeted segment resulted in growth phenotypes indistinguishable from those of strains expressing the native b subunit, suggesting abundant F1F0 ATP synthase activity. In contrast, several specific mutations were associated with a loss of enzyme function. Phagemids specifying the Ala-79----Pro, Arg-82----Pro, Arg-83----Pro, or Gln-85----Pro mutation failed to complement uncF (b) gene-deficient E. coli. F1F0 ATP synthase displayed the greatest sensitivity to mutations altering a single site in the target segment, Ala-79. The evidence suggests that Ala-79 occupies a restricted position in the enzyme complex.

Full text

PDF
7240

Images in this article

7243FIG. 2
on p.7243
7246FIG. 4
on p.7246

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aris J. P., Klionsky D. J., Simoni R. D. The Fo subunits of the Escherichia coli F1Fo-ATP synthase are sufficient to form a functional proton pore. J Biol Chem. 1985 Sep 15;260(20):11207–11215. [PubMed] [Google Scholar]
  2. Aris J. P., Simoni R. D. Cross-linking and labeling of the Escherichia coli F1F0-ATP synthase reveal a compact hydrophilic portion of F0 close to an F1 catalytic subunit. J Biol Chem. 1983 Dec 10;258(23):14599–14609. [PubMed] [Google Scholar]
  3. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cain B. D., Simoni R. D. Impaired proton conductivity resulting from mutations in the a subunit of F1F0 ATPase in Escherichia coli. J Biol Chem. 1986 Aug 5;261(22):10043–10050. [PubMed] [Google Scholar]
  5. Cain B. D., Simoni R. D. Interaction between Glu-219 and His-245 within the a subunit of F1F0-ATPase in Escherichia coli. J Biol Chem. 1988 May 15;263(14):6606–6612. [PubMed] [Google Scholar]
  6. Cain B. D., Simoni R. D. Proton translocation by the F1F0ATPase of Escherichia coli. Mutagenic analysis of the a subunit. J Biol Chem. 1989 Feb 25;264(6):3292–3300. [PubMed] [Google Scholar]
  7. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cox G. B., Fimmel A. L., Gibson F., Hatch L. The mechanism of ATP synthase: a reassessment of the functions of the b and a subunits. Biochim Biophys Acta. 1986 Apr 2;849(1):62–69. doi: 10.1016/0005-2728(86)90096-4. [DOI] [PubMed] [Google Scholar]
  9. Cox G. B., Jans D. A., Fimmel A. L., Gibson F., Hatch L. Hypothesis. The mechanism of ATP synthase. Conformational change by rotation of the beta-subunit. Biochim Biophys Acta. 1984 Dec 17;768(3-4):201–208. doi: 10.1016/0304-4173(84)90016-8. [DOI] [PubMed] [Google Scholar]
  10. Fillingame R. H., Peters L. K., White L. K., Mosher M. E., Paule C. R. Mutations altering aspartyl-61 of the omega subunit (uncE protein) of Escherichia coli H+ -ATPase differ in effect on coupled ATP hydrolysis. J Bacteriol. 1984 Jun;158(3):1078–1083. doi: 10.1128/jb.158.3.1078-1083.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fimmel A. L., Norris U. The F1F0-ATPase of Escherichia coli. The substitution of glycine by valine at position 29 in the c-subunit affects function but not assembly. Biochim Biophys Acta. 1989 Nov 27;986(2):257–262. doi: 10.1016/0005-2736(89)90475-6. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Futai M., Noumi T., Maeda M. ATP synthase (H+-ATPase): results by combined biochemical and molecular biological approaches. Annu Rev Biochem. 1989;58:111–136. doi: 10.1146/annurev.bi.58.070189.000551. [DOI] [PubMed] [Google Scholar]
  14. Gogol E. P., Lücken U., Capaldi R. A. The stalk connecting the F1 and F0 domains of ATP synthase visualized by electron microscopy of unstained specimens. FEBS Lett. 1987 Jul 27;219(2):274–278. doi: 10.1016/0014-5793(87)80234-x. [DOI] [PubMed] [Google Scholar]
  15. Hamilton C. M., Aldea M., Washburn B. K., Babitzke P., Kushner S. R. New method for generating deletions and gene replacements in Escherichia coli. J Bacteriol. 1989 Sep;171(9):4617–4622. doi: 10.1128/jb.171.9.4617-4622.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hermolin J., Gallant J., Fillingame R. H. Topology, organization, and function of the psi subunit in the F0 sector of the H+-ATPase of Escherichia coli. J Biol Chem. 1983 Dec 10;258(23):14550–14555. [PubMed] [Google Scholar]
  17. Hoppe J., Brunner J., Jørgensen B. B. Structure of the membrane-embedded F0 part of F1F0 ATP synthase from Escherichia coli as inferred from labeling with 3-(Trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. Biochemistry. 1984 Nov 6;23(23):5610–5616. doi: 10.1021/bi00318a035. [DOI] [PubMed] [Google Scholar]
  18. Hoppe J., Friedl P., Schairer H. U., Sebald W., von Meyenburg K., Jørgensen B. B. The topology of the proton translocating F0 component of the ATP synthase from E. coli K12: studies with proteases. EMBO J. 1983;2(1):105–110. doi: 10.1002/j.1460-2075.1983.tb01389.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Humbert R., Brusilow W. S., Gunsalus R. P., Klionsky D. J., Simoni R. D. Escherichia coli mutants defective in the uncH gene. J Bacteriol. 1983 Jan;153(1):416–422. doi: 10.1128/jb.153.1.416-422.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Klionsky D. J., Skalnik D. G., Simoni R. D. Differential translation of the genes encoding the proton-translocating ATPase of Escherichia coli. J Biol Chem. 1986 Jun 25;261(18):8096–8099. [PubMed] [Google Scholar]
  21. Kumamoto C. A., Simoni R. D. A mutation of the c subunit of the Escherichia coli proton-translocating ATPase that suppresses the effects of a mutant b subunit. J Biol Chem. 1987 Mar 5;262(7):3060–3064. [PubMed] [Google Scholar]
  22. Kumamoto C. A., Simoni R. D. Genetic evidence for interaction between the a and b subunits of the F0 portion of the Escherichia coli proton translocating ATPase. J Biol Chem. 1986 Aug 5;261(22):10037–10042. [PubMed] [Google Scholar]
  23. Lightowlers R. N., Howitt S. M., Hatch L., Gibson F., Cox G. B. The proton pore in the Escherichia coli F0F1-ATPase: a requirement for arginine at position 210 of the a-subunit. Biochim Biophys Acta. 1987 Dec 17;894(3):399–406. doi: 10.1016/0005-2728(87)90118-6. [DOI] [PubMed] [Google Scholar]
  24. Lücken U., Gogol E. P., Capaldi R. A. Structure of the ATP synthase complex (ECF1F0) of Escherichia coli from cryoelectron microscopy. Biochemistry. 1990 Jun 5;29(22):5339–5343. doi: 10.1021/bi00474a019. [DOI] [PubMed] [Google Scholar]
  25. McNeil J. B., Smith M. Saccharomyces cerevisiae CYC1 mRNA 5'-end positioning: analysis by in vitro mutagenesis, using synthetic duplexes with random mismatch base pairs. Mol Cell Biol. 1985 Dec;5(12):3545–3551. doi: 10.1128/mcb.5.12.3545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Nakamaye K. L., Eckstein F. Inhibition of restriction endonuclease Nci I cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis. Nucleic Acids Res. 1986 Dec 22;14(24):9679–9698. doi: 10.1093/nar/14.24.9679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Perlin D. S., Senior A. E. Functional effects and cross-reactivity of antibody to purified subunit b (uncF protein) of Escherichia coli proton-ATPase. Arch Biochem Biophys. 1985 Feb 1;236(2):603–611. doi: 10.1016/0003-9861(85)90664-2. [DOI] [PubMed] [Google Scholar]
  29. Porter A. C., Kumamoto C., Aldape K., Simoni R. D. Role of the b subunit of the Escherichia coli proton-translocating ATPase. A mutagenic analysis. J Biol Chem. 1985 Jul 5;260(13):8182–8187. [PubMed] [Google Scholar]
  30. Rose G. D., Gierasch L. M., Smith J. A. Turns in peptides and proteins. Adv Protein Chem. 1985;37:1–109. doi: 10.1016/s0065-3233(08)60063-7. [DOI] [PubMed] [Google Scholar]
  31. Russel M., Kidd S., Kelley M. R. An improved filamentous helper phage for generating single-stranded plasmid DNA. Gene. 1986;45(3):333–338. doi: 10.1016/0378-1119(86)90032-6. [DOI] [PubMed] [Google Scholar]
  32. Schaefer E. M., Hartz D., Gold L., Simoni R. D. Ribosome-binding sites and RNA-processing sites in the transcript of the Escherichia coli unc operon. J Bacteriol. 1989 Jul;171(7):3901–3908. doi: 10.1128/jb.171.7.3901-3908.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schneider E., Altendorf K. All three subunits are required for the reconstitution of an active proton channel (F0) of Escherichia coli ATP synthase (F1F0). EMBO J. 1985 Feb;4(2):515–518. doi: 10.1002/j.1460-2075.1985.tb03658.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Senior A. E. Secondary and tertiary structure of membrane proteins involved in proton translocation. Biochim Biophys Acta. 1983 Jul 15;726(2):81–95. doi: 10.1016/0304-4173(83)90001-0. [DOI] [PubMed] [Google Scholar]
  35. Senior A. E. The proton-translocating ATPase of Escherichia coli. Annu Rev Biophys Biophys Chem. 1990;19:7–41. doi: 10.1146/annurev.bb.19.060190.000255. [DOI] [PubMed] [Google Scholar]
  36. Steffens K., Schneider E., Deckers-Hebestreit G., Altendorf K. Fo portion of Escherichia coli ATP synthase. Further resolution of trypsin-generated fragments from subunit b. J Biol Chem. 1987 Apr 25;262(12):5866–5869. [PubMed] [Google Scholar]
  37. Takeyama M., Noumi T., Maeda M., Futai M. Fo portion of Escherichia coli H+-ATPase. Carboxyl-terminal region of the b subunit is essential for assembly of functional Fo. J Biol Chem. 1988 Nov 5;263(31):16106–16112. [PubMed] [Google Scholar]
  38. Walker J. E., Saraste M., Gay N. J. E. coli F1-ATPase interacts with a membrane protein component of a proton channel. Nature. 1982 Aug 26;298(5877):867–869. doi: 10.1038/298867a0. [DOI] [PubMed] [Google Scholar]
  39. 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]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES