Abstract
To study expression of uncG, the gene coding for the gamma subunit of the Escherichia coli proton-translocating ATPase, deletions were made in the intergenic region between uncA, the gene coding for the alpha subunit, and uncG. Two deletions which fused uncA and uncG coded for alpha-gamma fusion polypeptides which were synthesized well both in vitro and in vivo, demonstrating that uncG expression is normally controlled by nucleotides in the intergenic region. Multicopy plasmids carrying these fusion genes and the genes for the other subunits of the ATPase had a harmful effect on the growth of E. coli. The effect was overcome by N,N'-dicyclohexylcarbodiimide, indicating that the cells probably leaked protons. The deleterious effect was eliminated by making a nonpolar deletion in the upstream F0 gene uncB, or by cloning each of the uncA-uncG fusion genes onto a separate plasmid, removed from the F0 genes, thus demonstrating that the fusion genes were not primarily responsible for the proton permeability. A plasmid which carried F0 genes and the gene for the delta subunit caused deleterious proton leakiness in unc+ cells but not in cells from which the unc operon was deleted. The proton leakiness caused by these different plasmids was therefore due to the production of a leaky F0 proton channel and required the presence of F1 genes. The results support a model for ATPase assembly in which F1 genes or polypeptides are involved in the formation or opening of the F0 proton channel.
References
22
Referenced
21
-
Aris , J. , D. J. Klionsky , and R. D. Simoni . 1985 . The Fo subunits of the Escherichia coli F1Fo ATP-synthase are sufficient to form a functional proton pore . J. Biol. Chem. 260 : 11207 - 11215 .
(
10.1016/S0021-9258(17)39167-6
) / J. Biol. Chem. / The Fo subunits of the Escherichia coli F1Fo ATP-synthase are sufficient to form a functional proton pore by Aris J. (1985) -
Birnboim , H. C. , and J. Doly . 1979 . A rapid alkaline procedure for screening recombinant plasmid DNA . Nucleic Acids Res. 7 : 1513 - 1523 .
(
10.1093/nar/7.6.1513
) / Nucleic Acids Res. / A rapid alkaline procedure for screening recombinant plasmid DNA by Birnboim H. C. (1979) -
Brusilow , W. S. A. , R. P. Gunsalus , E. C. Hardeman , K. P. Decker , and R. D. Simoni . 1981 . In vitro synthesis of the Fo and F1 components of the proton translocating ATPase of Escherichia coli . J. Biol. Chem. 256 : 3141 - 3144 .
(
10.1016/S0021-9258(19)69576-1
) / J. Biol. Chem. / In vitro synthesis of the Fo and F1 components of the proton translocating ATPase of Escherichia coli by Brusilow W. S. A. (1981) -
Brusilow , W. S. A. , D. J. Klionsky , and R. D. Simoni . 1982 . Differential polypeptide synthesis of the proton-translocating ATPase of Escherichia coli . J. Bacteriol. 151 : 1363 - 1371 .
(
10.1128/jb.151.3.1363-1371.1982
) / J. Bacteriol. / Differential polypeptide synthesis of the proton-translocating ATPase of Escherichia coli by Brusilow W. S. A. (1982) -
Chang , A. C. Y. , and S. N. Cohen . 1978 . Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid . J. Bacteriol. 134 : 1141 - 1156 .
(
10.1128/jb.134.3.1141-1156.1978
) / J. Bacteriol. / Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid by Chang A. C. Y. (1978) -
Cox , G. B. , J. A. Downie , L. Langman , A. E. Senior , G. Ash , D. R. H. Fayle , and F. Gibson . 1981 . Assembly of the adenosine triphophatase complex in Escherichia coli: assembly of Fo is dependent on the formation of specific F1 subunits . J. Bacteriol. 148 : 30 - 42 .
(
10.1128/jb.148.1.30-42.1981
) / J. Bacteriol. / Assembly of the adenosine triphophatase complex in Escherichia coli: assembly of Fo is dependent on the formation of specific F1 subunits by Cox G. B. (1981) -
Downie , J. A. , L. Langman , G. B. Cox , C. Yanofsky , and F. Gibson . 1980 . Subunits of the adenosine triphosphatase complex translated in vitro from the Escherichia coli unc operon . J. Bacteriol. 143 : 8 - 17 .
(
10.1128/jb.143.1.8-17.1980
) / J. Bacteriol. / Subunits of the adenosine triphosphatase complex translated in vitro from the Escherichia coli unc operon by Downie J. A. (1980) -
Fillingame , R. H. , B. Porter , J. Hermolin , and L. K. White . 1986 . Synthesis of a functional Fo sector of the Escherichia coli H+-ATPase does not require synthesis of the alpha or beta subunits of F1 . J. Bacteriol. 164 : 244 - 251 .
(
10.1128/jb.165.1.244-251.1986
) / J. Bacteriol. / Synthesis of a functional Fo sector of the Escherichia coli H+-ATPase does not require synthesis of the alpha or beta subunits of F1 by Fillingame R. H. (1986) -
Foster , D. L. , and R. H. Fillingame . 1982 . Stoichiometry of subunits in the H+-ATPase complex of Escherichia coli . J. Biol. Chem. 257 : 2009 - 2015 .
(
10.1016/S0021-9258(19)68140-8
) / J. Biol. Chem. / Stoichiometry of subunits in the H+-ATPase complex of Escherichia coli by Foster D. L. (1982) -
Futai , M. , and H. Kanazawa . 1983 . Structure and function of proton-translocating adenosine triphosphatase (FoF1): biochemical and molecular biological approaches . Microbiol. Rev. 47 : 285 - 312 .
(
10.1128/mr.47.3.285-312.1983
) / Microbiol. Rev. / Structure and function of proton-translocating adenosine triphosphatase (FoF1): biochemical and molecular biological approaches by Futai M. (1983) -
Gunsalus , R. P. , W. S. A. Brusilow , and R. D. Simoni . 1982 . Gene order and gene-polypeptide relationships of the protontranslocating ATPase operon (unc) of Escherichia coli . Proc. Natl. Acad. Sci. USA 79 : 320 - 324 .
(
10.1073/pnas.79.2.320
) / Proc. Natl. Acad. Sci. USA / Gene order and gene-polypeptide relationships of the protontranslocating ATPase operon (unc) of Escherichia coli by Gunsalus R. P. (1982) -
Humbert , R. , W. S. A. Brusilow , R. P. Gunsalus , D. J. Klionsky , and R. D. Simoni . 1983 . Escherichia coli mutants defective in the uncH gene . J. Bacteriol. 153 : 416 - 422 .
(
10.1128/jb.153.1.416-422.1983
) / J. Bacteriol. / Escherichia coli mutants defective in the uncH gene by Humbert R. (1983) -
Jones , H. M. , C. M. Brajkovich , and R. P. Gunsalus . 1983 . In vivo 5'-terminus and length of the mRNA for the protontranslocating ATPase (unc) operon of Escherichia coli . J. Bacteriol. 155 : 1279 - 1287 .
(
10.1128/jb.155.3.1279-1287.1983
) / J. Bacteriol. / In vivo 5'-terminus and length of the mRNA for the protontranslocating ATPase (unc) operon of Escherichia coli by Jones H. M. (1983) -
Kanazawa , H. , T. Kayano , K. Mabuchi , and M. Futai . 1981 . Nucleotide sequence of the genes coding for the a, P, and y subunits of the proton-translocating ATPase of Escherichia coli . Biochem. Biophys. Res. Commun. 103 : 604 - 612 .
(
10.1016/0006-291X(81)90494-0
) / Biochem. Biophys. Res. Commun. / Nucleotide sequence of the genes coding for the a, P, and y subunits of the proton-translocating ATPase of Escherichia coli by Kanazawa H. (1981) - Klionsky , D. J. , W. S. A. Brusilow , and R. D. Simoni . 1983 . Assembly of a functional Fo of the proton-translocating ATPase of Escherichia coli . J. Bacteriol. 258 : 10136 - 10143 . / J. Bacteriol. / Assembly of a functional Fo of the proton-translocating ATPase of Escherichia coli by Klionsky D. J. (1983)
-
Laemmli , U. K. 1970 . Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . Nature (London) 227 : 680 - 685 .
(
10.1038/227680a0
) / Nature (London) / Cleavage of structural proteins during the assembly of the head of bacteriophage T4 by Laemmli U. K. (1970) - Miller J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory Cold Spring Harbor N.Y.
-
Nielsen , J. , F. G. Hansen , J. Hoppe , P. Friedl , and K. von Meyenberg . 1981 . The nucleotide sequence of the atp genes coding for the Fo subunits a, b, c, and the F1 subunit 8 of the membrane bound ATP synthase of Escherichia coli . Mol. Gen. Genet. 184 : 33 - 39 .
(
10.1007/BF00271191
) / Mol. Gen. Genet. / The nucleotide sequence of the atp genes coding for the Fo subunits a, b, c, and the F1 subunit 8 of the membrane bound ATP synthase of Escherichia coli by Nielsen J. (1981) -
Sanger , F. , A. R. Coulson , B. G. Barrel , A. J. H. Smithe , and B. A. Row . 1980 . Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing . J. Mol. Biol. 143 : 161 - 178 .
(
10.1016/0022-2836(80)90196-5
) / J. Mol. Biol. / Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing by Sanger F. (1980) -
Sebald , W. , and J. Hoppe . 1981 . On the structure and genetics of the proteolipid subunit of the ATP synthase complex . Curr. Top. Bioenerg. 12 : 1 - 64 .
(
10.1016/B978-0-12-152512-5.50007-5
) / Curr. Top. Bioenerg. / On the structure and genetics of the proteolipid subunit of the ATP synthase complex by Sebald W. (1981) -
Vieira , J. , and J. Messing . 1982 . The pUC plasmids, an M13mp 7-derived system for insertion mutagenesis and sequencing with synthetic primers . Gene 19 : 259 - 268 .
(
10.1016/0378-1119(82)90015-4
) / Gene / The pUC plasmids, an M13mp 7-derived system for insertion mutagenesis and sequencing with synthetic primers by Vieira J. (1982) - Walker , J. E. , M. Saraste , and N. J. Gay . 1984 . The unc operon-nucleotide sequence, regulation, and structure of ATPsynthase . Biochem. Biophys. Acta 768 : 164 - 200 . / Biochem. Biophys. Acta / The unc operon-nucleotide sequence, regulation, and structure of ATPsynthase by Walker J. E. (1984)
Dates
Type | When |
---|---|
Created | 8 years, 9 months ago (Nov. 2, 2016, 3:17 p.m.) |
Deposited | 4 years ago (July 29, 2021, 1:43 p.m.) |
Indexed | 1 month, 4 weeks ago (June 27, 2025, 6:38 a.m.) |
Issued | 37 years, 9 months ago (Nov. 1, 1987) |
Published | 37 years, 9 months ago (Nov. 1, 1987) |
Published Print | 37 years, 9 months ago (Nov. 1, 1987) |
@article{Brusilow_1987, title={Proton leakiness caused by cloned genes for the F0 sector of the proton-translocating ATPase of Escherichia coli: requirement for F1 genes}, volume={169}, ISSN={1098-5530}, url={http://dx.doi.org/10.1128/jb.169.11.4984-4990.1987}, DOI={10.1128/jb.169.11.4984-4990.1987}, number={11}, journal={Journal of Bacteriology}, publisher={American Society for Microbiology}, author={Brusilow, W S}, year={1987}, month=nov, pages={4984–4990} }