Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Feb;179(3):735–741. doi: 10.1128/jb.179.3.735-741.1997

Role of conserved residues in hydrophilic loop 8-9 of the lactose permease.

N J Pazdernik 1, A E Jessen-Marshall 1, R J Brooker 1
PMCID: PMC178755  PMID: 9006028

Abstract

A peptide motif, GXXX(D/E)(R/K)XG(R/K)(R/K), has been conserved in a large group of evolutionarily related membrane proteins that transport small molecules across the membrane. Within the superfamily, this motif is located in two cytoplasmic loops that connect transmembrane segments 2 and 3 and transmembrane segments 8 and 9. In a previous study concerning the loop 2-3 motif of the lactose permease (A. E. Jessen-Marshall, N. J. Paul, and R. J. Brooker, J. Biol. Chem. 270:16251-16257, 1995), it was shown that the first-position glycine and the fifth-position aspartate are critical for transport activity since a variety of site-directed mutations greatly diminished the rate of transport. In the current study, a similar approach was used to investigate the functional significance of the conserved residues in the loop 8-9 motif. In the wild-type lactose permease, however, this motif has been evolutionarily modified so that the first-position glycine (an alpha-helix breaker) has been changed to proline (also a helix breaker); the fifth position has been changed to an asparagine; and one of the basic residues has been altered. In this investigation, we made a total of 28 single and 7 double mutants within the loop 8-9 motif to explore the functional importance of this loop. With regard to transport activity, amino acid substitutions within the loop 8-9 motif tend to be fairly well tolerated. Most substitutions produced permeases with normal or mildly defective transport activities. However, three substitutions at the first position (i.e., position 280) resulted in defective lactose transport. Kinetic analysis of position 280 mutants indicated that the defect decreased the Vmax for lactose uptake. Besides substitutions at position 280, a Gly-288-to-Thr mutant had the interesting property that the kinetic parameters for lactose uptake were normal yet the rates of lactose efflux and exchange were approximately 10-fold faster than wild-type rates. The results of this study suggest that loop 8-9 may facilitate conformational changes that translocate lactose.

Full Text

The Full Text of this article is available as a PDF (220.9 KB).

Selected References

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

  1. BUTTIN G., COHEN G. N., MONOD J., RICKENBERG H. V. La galactoside-perméase d'Escherichia coli. Ann Inst Pasteur (Paris) 1956 Dec;91(6):829–857. [PubMed] [Google Scholar]
  2. 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]
  3. Brooker R. J. An analysis of lactose permease "sugar specificity" mutations which also affect the coupling between proton and lactose transport. I. Val177 and Val177/Asn319 permeases facilitate proton uniport and sugar uniport. J Biol Chem. 1991 Mar 5;266(7):4131–4138. [PubMed] [Google Scholar]
  4. Brooker R. J. The lactose permease of Escherichia coli. Res Microbiol. 1990 Mar-Apr;141(3):309–315. doi: 10.1016/0923-2508(90)90004-a. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. Calamia J., Manoil C. lac permease of Escherichia coli: topology and sequence elements promoting membrane insertion. Proc Natl Acad Sci U S A. 1990 Jul;87(13):4937–4941. doi: 10.1073/pnas.87.13.4937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Crane R. K. The gradient hypothesis and other models of carrier-mediated active transport. Rev Physiol Biochem Pharmacol. 1977;78:99–159. doi: 10.1007/BFb0027722. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Franco P. J., Brooker R. J. Functional roles of Glu-269 and Glu-325 within the lactose permease of Escherichia coli. J Biol Chem. 1994 Mar 11;269(10):7379–7386. [PubMed] [Google Scholar]
  12. Goswitz V. C., Brooker R. J. Structural features of the uniporter/symporter/antiporter superfamily. Protein Sci. 1995 Mar;4(3):534–537. doi: 10.1002/pro.5560040319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Griffith J. K., Baker M. E., Rouch D. A., Page M. G., Skurray R. A., Paulsen I. T., Chater K. F., Baldwin S. A., Henderson P. J. Membrane transport proteins: implications of sequence comparisons. Curr Opin Cell Biol. 1992 Aug;4(4):684–695. doi: 10.1016/0955-0674(92)90090-y. [DOI] [PubMed] [Google Scholar]
  14. Henderson P. J. Proton-linked sugar transport systems in bacteria. J Bioenerg Biomembr. 1990 Aug;22(4):525–569. doi: 10.1007/BF00762961. [DOI] [PubMed] [Google Scholar]
  15. Jessen-Marshall A. E., Paul N. J., Brooker R. J. The conserved motif, GXXX(D/E)(R/K)XG[X](R/K)(R/K), in hydrophilic loop 2/3 of the lactose permease. J Biol Chem. 1995 Jul 7;270(27):16251–16257. doi: 10.1074/jbc.270.27.16251. [DOI] [PubMed] [Google Scholar]
  16. Jung K., Jung H., Colacurcio P., Kaback H. R. Role of glycine residues in the structure and function of lactose permease, an Escherichia coli membrane transport protein. Biochemistry. 1995 Jan 24;34(3):1030–1039. doi: 10.1021/bi00003a038. [DOI] [PubMed] [Google Scholar]
  17. Kaczorowski G. J., Kaback H. R. Mechanism of lactose translocation in membrane vesicles from Escherichia coli. 1. Effect of pH on efflux, exchange, and counterflow. Biochemistry. 1979 Aug 21;18(17):3691–3697. doi: 10.1021/bi00584a009. [DOI] [PubMed] [Google Scholar]
  18. Kaczorowski G. J., Robertson D. E., Kaback H. R. Mechanism of lactose translocation in membrane vesicles from Escherichia coli. 2. Effect of imposed delata psi, delta pH, and Delta mu H+. Biochemistry. 1979 Aug 21;18(17):3697–3704. doi: 10.1021/bi00584a010. [DOI] [PubMed] [Google Scholar]
  19. King S. C., Hansen C. L., Wilson T. H. The interaction between aspartic acid 237 and lysine 358 in the lactose carrier of Escherichia coli. Biochim Biophys Acta. 1991 Feb 25;1062(2):177–186. doi: 10.1016/0005-2736(91)90390-t. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  22. Maiden M. C., Davis E. O., Baldwin S. A., Moore D. C., Henderson P. J. Mammalian and bacterial sugar transport proteins are homologous. Nature. 1987 Feb 12;325(6105):641–643. doi: 10.1038/325641a0. [DOI] [PubMed] [Google Scholar]
  23. Marger M. D., Saier M. H., Jr A major superfamily of transmembrane facilitators that catalyse uniport, symport and antiport. Trends Biochem Sci. 1993 Jan;18(1):13–20. doi: 10.1016/0968-0004(93)90081-w. [DOI] [PubMed] [Google Scholar]
  24. McKenna E., Hardy D., Kaback H. R. Insertional mutagenesis of hydrophilic domains in the lactose permease of Escherichia coli. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):11954–11958. doi: 10.1073/pnas.89.24.11954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Newman M. J., Wilson T. H. Solubilization and reconstitution of the lactose transport system from Escherichia coli. J Biol Chem. 1980 Nov 25;255(22):10583–10586. [PubMed] [Google Scholar]
  26. Roepe P. D., Consler T. G., Menezes M. E., Kaback H. R. The lac permease of Escherichia coli: site-directed mutagenesis studies on the mechanism of beta-galactoside/H+ symport. Res Microbiol. 1990 Mar-Apr;141(3):290–308. doi: 10.1016/0923-2508(90)90003-9. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. 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]
  30. West I. C., Mitchell P. Stoicheiometry of lactose-H+ symport across the plasma membrane of Escherichia coli. Biochem J. 1973 Mar;132(3):587–592. doi: 10.1042/bj1320587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Yamaguchi A., Kimura T., Sawai T. Hot spots for sulfhydryl inactivation of Cys mutants in the widely conserved sequence motifs of the metal-tetracycline/H+ antiporter of Escherichia coli. J Biochem. 1994 May;115(5):958–964. doi: 10.1093/oxfordjournals.jbchem.a124445. [DOI] [PubMed] [Google Scholar]
  32. Yamaguchi A., Kimura T., Someya Y., Sawai T. Metal-tetracycline/H+ antiporter of Escherichia coli encoded by transposon Tn10. The structural resemblance and functional difference in the role of the duplicated sequence motif between hydrophobic segments 2 and 3 and segments 8 and 9. J Biol Chem. 1993 Mar 25;268(9):6496–6504. [PubMed] [Google Scholar]
  33. Yamaguchi A., Ono N., Akasaka T., Noumi T., Sawai T. Metal-tetracycline/H+ antiporter of Escherichia coli encoded by a transposon, Tn10. The role of the conserved dipeptide, Ser65-Asp66, in tetracycline transport. J Biol Chem. 1990 Sep 15;265(26):15525–15530. [PubMed] [Google Scholar]
  34. Yamaguchi A., Someya Y., Sawai T. Metal-tetracycline/H+ antiporter of Escherichia coli encoded by transposon Tn10. The role of a conserved sequence motif, GXXXXRXGRR, in a putative cytoplasmic loop between helices 2 and 3. J Biol Chem. 1992 Sep 25;267(27):19155–19162. [PubMed] [Google Scholar]
  35. 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]
  36. 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 Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES