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. 1987 Aug;84(16):5535–5539. doi: 10.1073/pnas.84.16.5535

Lactose permease of Escherichia coli: properties of mutants defective in substrate translocation.

P Overath, U Weigel, J M Neuhaus, J Soppa, R Seckler, I Riede, H Bocklage, B Müller-Hill, G Aichele, J K Wright
PMCID: PMC298897  PMID: 3303027

Abstract

Mutants of lactose permease of Escherichia coli with amino acid changes (Gly-24----Glu; Gly-24----Arg; Pro-28---Ser; Gly-24, Pro-28----Glu-Ser and Gly-24, Pro-28----Arg-Ser) within a putative membrane-spanning alpha-helix (Phe-Gly-Leu-Phe-Phe-Phe-Phe-Tyr-Phe-Phe-Ile-Met-Gly- Ala-Tyr-Phe-Pro-Phe-Phe-Pro-Ile) are incorporated into the cytoplasmic membrane. The mutant proteins retain the ability to bind galactosides, and the affinity for several substrates is actually increased. However, the rate of active transport is decreased to 0.01% of the wild-type rate in the mutants carrying Arg-24 or Arg-24, Ser-28. Kinetic analysis demonstrates that the two mutants require 10 min to cause occupied binding sites for galactoside and H+ to change their exposure from the periplasm to the cytoplasm as compared to 50 ms in the wild type. The effect is less pronounced when these sites are unoccupied.

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Selected References

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

  1. Bieseler B., Prinz H., Beyreuther K. Topological studies of lactose permease of Escherichia coli by protein sequence analysis. Ann N Y Acad Sci. 1985;456:309–325. doi: 10.1111/j.1749-6632.1985.tb14882.x. [DOI] [PubMed] [Google Scholar]
  2. Brooker R. J., Wilson T. H. Isolation and nucleotide sequencing of lactose carrier mutants that transport maltose. Proc Natl Acad Sci U S A. 1985 Jun;82(12):3959–3963. doi: 10.1073/pnas.82.12.3959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Dornmair K., Corin A. F., Wright J. K., Jähnig F. The size of the lactose permease derived from rotational diffusion measurements. EMBO J. 1985 Dec 16;4(13A):3633–3638. doi: 10.1002/j.1460-2075.1985.tb04127.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ehring R., Beyreuther K., Wright J. K., Overath P. In vitro and in vivo products of E. coli lactose permease gene are identical. Nature. 1980 Feb 7;283(5747):537–540. doi: 10.1038/283537a0. [DOI] [PubMed] [Google Scholar]
  7. Hobson A. C., Gho D., Müller-Hill B. Isolation, genetic analysis, and characterization of Escherichia coli mutants with defects in the lacY gene. J Bacteriol. 1977 Sep;131(3):830–838. doi: 10.1128/jb.131.3.830-838.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Humphreys G. O., Willshaw G. A., Smith H. R., Anderson E. S. Mutagenesis of plasmid DNA with hydroxylamine: isolation of mutants of multi-copy plasmids. Mol Gen Genet. 1976 Apr 23;145(1):101–108. doi: 10.1007/BF00331564. [DOI] [PubMed] [Google Scholar]
  9. Jähnig F. Thermodynamics and kinetics of protein incorporation into membranes. Proc Natl Acad Sci U S A. 1983 Jun;80(12):3691–3695. doi: 10.1073/pnas.80.12.3691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kaback H. R. Active transport in Escherichia coli: passage to permease. Annu Rev Biophys Biophys Chem. 1986;15:279–319. doi: 10.1146/annurev.bb.15.060186.001431. [DOI] [PubMed] [Google Scholar]
  11. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Markgraf M., Bocklage H., Müller-Hill B. A change of threonine 266 to isoleucine in the lac permease of Escherichia coli diminishes the transport of lactose and increases the transport of maltose. Mol Gen Genet. 1985;198(3):473–475. doi: 10.1007/BF00332941. [DOI] [PubMed] [Google Scholar]
  13. Mitaku S., Wright J. K., Best L., Jähnig F. Localization of the galactoside binding site in the lactose carrier of Escherichia coli. Biochim Biophys Acta. 1984 Oct 3;776(2):247–258. doi: 10.1016/0005-2736(84)90214-1. [DOI] [PubMed] [Google Scholar]
  14. Nelson S. O., Wright J. K., Postma P. W. The mechanism of inducer exclusion. Direct interaction between purified III of the phosphoenolpyruvate:sugar phosphotransferase system and the lactose carrier of Escherichia coli. EMBO J. 1983;2(5):715–720. doi: 10.1002/j.1460-2075.1983.tb01490.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Oliver D. Protein secretion in Escherichia coli. Annu Rev Microbiol. 1985;39:615–648. doi: 10.1146/annurev.mi.39.100185.003151. [DOI] [PubMed] [Google Scholar]
  16. Seckler R., Möröy T., Wright J. K., Overath P. Anti-peptide antibodies and proteases as structural probes for the lactose/H+ transporter of Escherichia coli: a loop around amino acid residue 130 faces the cytoplasmic side of the membrane. Biochemistry. 1986 May 6;25(9):2403–2409. doi: 10.1021/bi00357a016. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Seckler R., Wright J. K. Sidedness of native membrane vesicles of Escherichia coli and orientation of the reconstituted lactose: H+ carrier. Eur J Biochem. 1984 Jul 16;142(2):269–279. doi: 10.1111/j.1432-1033.1984.tb08281.x. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Vogel H., Wright J. K., Jähnig F. The structure of the lactose permease derived from Raman spectroscopy and prediction methods. EMBO J. 1985 Dec 16;4(13A):3625–3631. doi: 10.1002/j.1460-2075.1985.tb04126.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Wickner W. T., Lodish H. F. Multiple mechanisms of protein insertion into and across membranes. Science. 1985 Oct 25;230(4724):400–407. doi: 10.1126/science.4048938. [DOI] [PubMed] [Google Scholar]
  22. Wright J. K., Dornmair K., Mitaku S., Möröy T., Neuhaus J. M., Seckler R., Vogel H., Weigel U., Jähnig F., Overath P. Lactose: H+ carrier of Escherichia coli: kinetic mechanism, purification, and structure. Ann N Y Acad Sci. 1985;456:326–341. doi: 10.1111/j.1749-6632.1985.tb14883.x. [DOI] [PubMed] [Google Scholar]
  23. Wright J. K., Riede I., Overath P. Lactose carrier protein of Escherichia coli: interaction with galactosides and protons. Biochemistry. 1981 Oct 27;20(22):6404–6415. doi: 10.1021/bi00525a019. [DOI] [PubMed] [Google Scholar]
  24. Wright J. K., Seckler R., Overath P. Molecular aspects of sugar:ion cotransport. Annu Rev Biochem. 1986;55:225–248. doi: 10.1146/annurev.bi.55.070186.001301. [DOI] [PubMed] [Google Scholar]
  25. Wright J. K., Seckler R. The lactose/H+ carrier of Escherichia coli: lac YUN mutation decreases the rate of active transport and mimics an energy-uncoupled phenotype. Biochem J. 1985 Apr 1;227(1):287–297. doi: 10.1042/bj2270287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Wright J. K., Teather R. M., Overath P. Lactose permease of Escherichia coli. Methods Enzymol. 1983;97:158–175. doi: 10.1016/0076-6879(83)97130-6. [DOI] [PubMed] [Google Scholar]
  27. Wright J. K. The kinetic mechanism of galactoside/H+ cotransport in Escherichia coli. Biochim Biophys Acta. 1986 Mar 13;855(3):391–416. doi: 10.1016/0005-2736(86)90085-4. [DOI] [PubMed] [Google Scholar]
  28. Wright J. K., Weigel U., Lustig A., Bocklage H., Mieschendahl M., Müller-Hill B., Overath P. Does the lactose carrier of Escherichia coli function as a monomer? FEBS Lett. 1983 Oct 3;162(1):11–15. doi: 10.1016/0014-5793(83)81039-4. [DOI] [PubMed] [Google Scholar]
  29. von Heijne G. Signal sequences. The limits of variation. J Mol Biol. 1985 Jul 5;184(1):99–105. doi: 10.1016/0022-2836(85)90046-4. [DOI] [PubMed] [Google Scholar]

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