Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1985 Oct;82(20):6765–6768. doi: 10.1073/pnas.82.20.6765

Site-specific mutagenesis of histidine residues in the lac permease of Escherichia coli.

E Padan, H K Sarkar, P V Viitanen, M S Poonian, H R Kaback
PMCID: PMC390767  PMID: 3901007

Abstract

The lacY gene of Escherichia coli, which encodes the lac permease, has been modified by oligonucleotide-directed, site-specific mutagenesis such that each of the four histidine residues in the molecule is replaced with an arginine residue. Replacement of histidine-35 and histidine-39 with arginine has no apparent effect on permease activity. In contrast, replacement of either histidine-205 or histidine-322 by arginine causes a dramatic loss of transport activity, although the cells contain a normal complement of permease molecules, as determined by immunoadsorption assays. Interestingly, although substitution of histidine-205 or histidine-322 by arginine results in the loss of ability to catalyze active lactose transport, permease molecules with arginine at residue 322 appear to facilitate downhill lactose movements at high concentrations of the disaccharide. The results provide strong support for the contention that histidine residues in the lac permease play an important role in the coupling between lactose and proton translocation.

Full text

PDF
6768

Images in this article

6766Image
on p.6766
6766Image
on p.6766
6766Image
on p.6766
6767Image
on p.6767

Selected References

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

  1. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  2. Büchel D. E., Gronenborn B., Müller-Hill B. Sequence of the lactose permease gene. Nature. 1980 Feb 7;283(5747):541–545. doi: 10.1038/283541a0. [DOI] [PubMed] [Google Scholar]
  3. Carrasco N., Herzlinger D., Mitchell R., DeChiara S., Danho W., Gabriel T. F., Kaback H. R. Intramolecular dislocation of the COOH terminus of the lac carrier protein in reconstituted proteoliposomes. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4672–4676. doi: 10.1073/pnas.81.15.4672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Carrasco N., Tahara S. M., Patel L., Goldkorn T., Kaback H. R. Preparation, characterization, and properties of monoclonal antibodies against the lac carrier protein from Escherichia coli. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6894–6898. doi: 10.1073/pnas.79.22.6894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carrasco N., Viitanen P., Herzlinger D., Kaback H. R. Monoclonal antibodies against the lac carrier protein from Escherichia coli. 1. Functional studies. Biochemistry. 1984 Jul 31;23(16):3681–3687. doi: 10.1021/bi00311a017. [DOI] [PubMed] [Google Scholar]
  6. Costello M. J., Viitanen P., Carrasco N., Foster D. L., Kaback H. R. Morphology of proteoliposomes reconstituted with purified lac carrier protein from Escherichia coli. J Biol Chem. 1984 Dec 25;259(24):15579–15586. [PubMed] [Google Scholar]
  7. Foster D. L., Boublik M., Kaback H. R. Structure of the lac carrier protein of Escherichia coli. J Biol Chem. 1983 Jan 10;258(1):31–34. [PubMed] [Google Scholar]
  8. Foster D. L., Garcia M. L., Newman M. J., Patel L., Kaback H. R. Lactose-proton symport by purified lac carrier protein. Biochemistry. 1982 Oct 26;21(22):5634–5638. doi: 10.1021/bi00265a038. [DOI] [PubMed] [Google Scholar]
  9. Garcia M. L., Patel L., Padan E., Kaback H. R. Mechanism of lactose transport in Escherichia coli membrane vesicles: evidence for the involvement of histidine residue(s) in the response of the lac carrier to the proton electrochemical gradient. Biochemistry. 1982 Nov 9;21(23):5800–5805. doi: 10.1021/bi00266a012. [DOI] [PubMed] [Google Scholar]
  10. Garcia M. L., Viitanen P., Foster D. L., Kaback H. R. Mechanism of lactose translocation in proteoliposomes reconstituted with lac carrier protein purified from Escherichia coli. 1. Effect of pH and imposed membrane potential on efflux, exchange, and counterflow. Biochemistry. 1983 May 10;22(10):2524–2531. doi: 10.1021/bi00279a033. [DOI] [PubMed] [Google Scholar]
  11. Goldkorn T., Rimon G., Kaback H. R. Topology of the lac carrier protein in the membrane of Escherichia coli. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3322–3326. doi: 10.1073/pnas.80.11.3322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Herzlinger D., Carrasco N., Kaback H. R. Functional and immunochemical characterization of a mutant of Escherichia coli energy uncoupled for lactose transport. Biochemistry. 1985 Jan 1;24(1):221–229. doi: 10.1021/bi00322a032. [DOI] [PubMed] [Google Scholar]
  13. Herzlinger D., Viitanen P., Carrasco N., Kaback H. R. Monoclonal antibodies against the lac carrier protein from Escherichia coli. 2. Binding studies with membrane vesicles and proteoliposomes reconstituted with purified lac carrier protein. Biochemistry. 1984 Jul 31;23(16):3688–3693. doi: 10.1021/bi00311a018. [DOI] [PubMed] [Google Scholar]
  14. Kaback H. R. The lac carrier protein in Escherichia coli. J Membr Biol. 1983;76(2):95–112. doi: 10.1007/BF02000610. [DOI] [PubMed] [Google Scholar]
  15. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  16. Matsushita K., Patel L., Gennis R. B., Kaback H. R. Reconstitution of active transport in proteoliposomes containing cytochrome o oxidase and lac carrier protein purified from Escherichia coli. Proc Natl Acad Sci U S A. 1983 Aug;80(16):4889–4893. doi: 10.1073/pnas.80.16.4889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  18. Newman M. J., Foster D. L., Wilson T. H., Kaback H. R. Purification and reconstitution of functional lactose carrier from Escherichia coli. J Biol Chem. 1981 Nov 25;256(22):11804–11808. [PubMed] [Google Scholar]
  19. Padan E., Patel L., Kaback H. R. Effect of diethylpyrocarbonate on lactose/proton symport in Escherichia coli membrane vesicles. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6221–6225. doi: 10.1073/pnas.76.12.6221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Patel L., Garcia M. L., Kaback H. R. Direct measurement of lactose/proton symport in Escherichia coli membrane vesicles: further evidence for the involvement of histidine residue(s). Biochemistry. 1982 Nov 9;21(23):5805–5810. doi: 10.1021/bi00266a013. [DOI] [PubMed] [Google Scholar]
  21. Sanger F., Coulson A. R. The use of thin acrylamide gels for DNA sequencing. FEBS Lett. 1978 Mar 1;87(1):107–110. doi: 10.1016/0014-5793(78)80145-8. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Seckler R., Wright J. K., Overath P. Peptide-specific antibody locates the COOH terminus of the lactose carrier of Escherichia coli on the cytoplasmic side of the plasma membrane. J Biol Chem. 1983 Sep 25;258(18):10817–10820. [PubMed] [Google Scholar]
  24. Teather R. M., Bramhall J., Riede I., Wright J. K., Fürst M., Aichele G., Wilhelm U., Overath P. Lactose carrier protein of Escherichia coli. Structure and expression of plasmids carrying the Y gene of the lac operon. Eur J Biochem. 1980;108(1):223–231. doi: 10.1111/j.1432-1033.1980.tb04715.x. [DOI] [PubMed] [Google Scholar]
  25. Teather R. M., Müller-Hill B., Abrutsch U., Aichele G., Overath P. Amplification of the lactose carrier protein in Escherichia coli using a plasmid vector. Mol Gen Genet. 1978 Feb 27;159(3):239–248. doi: 10.1007/BF00268260. [DOI] [PubMed] [Google Scholar]
  26. Trumble W. R., Viitanen P. V., Sarkar H. K., Poonian M. S., Kaback H. R. Site-directed mutagenesis of cys148 in the lac carrier protein of Escherichia coli. Biochem Biophys Res Commun. 1984 Mar 30;119(3):860–867. doi: 10.1016/0006-291x(84)90853-2. [DOI] [PubMed] [Google Scholar]
  27. Viitanen P., Garcia M. L., Foster D. L., Kaczorowski G. J., Kaback H. R. Mechanism of lactose translocation in proteoliposomes reconstituted with lac carrier protein purified from Escherichia coli. 2. Deuterium solvent isotope effects. Biochemistry. 1983 May 10;22(10):2531–2536. doi: 10.1021/bi00279a034. [DOI] [PubMed] [Google Scholar]
  28. Viitanen P., Garcia M. L., Kaback H. R. Purified reconstituted lac carrier protein from Escherichia coli is fully functional. Proc Natl Acad Sci U S A. 1984 Mar;81(6):1629–1633. doi: 10.1073/pnas.81.6.1629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wright J. K., Overath P. Purification of the lactose:H+ carrier of Escherichia coli and characterization of galactoside binding and transport. Eur J Biochem. 1984 Feb 1;138(3):497–508. doi: 10.1111/j.1432-1033.1984.tb07944.x. [DOI] [PubMed] [Google Scholar]
  30. Zilberstein D., Agmon V., Schuldiner S., Padan E. The sodium/proton antiporter is part of the pH homeostasis mechanism in Escherichia coli. J Biol Chem. 1982 Apr 10;257(7):3687–3691. [PubMed] [Google Scholar]
  31. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 1983;100:468–500. doi: 10.1016/0076-6879(83)00074-9. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES