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. 1996 May;178(9):2650–2655. doi: 10.1128/jb.178.9.2650-2655.1996

Topology of the phenylalanine-specific permease of Escherichia coli.

J Pi 1, A J Pittard 1
PMCID: PMC177991  PMID: 8626334

Abstract

The PheP protein is a high-affinity phenylalanine-specific permease of the bacterium Escherichia coli. A topological model based on sequence analysis of the putative protein in which PheP has 12 transmembrane segments with both N and C termini located in the cytoplasm had been proposed (J. Pi, P. J. Wookey, and A. J. Pittard, J. Bacteriol. 173:3622-3629, 1991). This topological model of PheP has been further examined by generating protein fusions with alkaline phosphatase. Twenty-five sandwich fusion proteins have been constructed by inserting the 'phoA gene at specific sites within the pheP gene. In general, the PhoA activities of the fusions support a PheP topology model consisting of 12 transmembrane segments with the N and C termini in the cytoplasm. However, alterations to the model, affecting spans III and VI, were indicated by this analysis and were supported by additional site-directed mutagenesis of some of the residues involved.

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Selected References

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  1. Bachmann B. J. Linkage map of Escherichia coli K-12, edition 8. Microbiol Rev. 1990 Jun;54(2):130–197. doi: 10.1128/mr.54.2.130-197.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bibi E., Béjà O. Membrane topology of multidrug resistance protein expressed in Escherichia coli. N-terminal domain. J Biol Chem. 1994 Aug 5;269(31):19910–19915. [PubMed] [Google Scholar]
  3. Brickman E., Beckwith J. Analysis of the regulation of Escherichia coli alkaline phosphatase synthesis using deletions and phi80 transducing phages. J Mol Biol. 1975 Aug 5;96(2):307–316. doi: 10.1016/0022-2836(75)90350-2. [DOI] [PubMed] [Google Scholar]
  4. Chou P. Y., Fasman G. D. Empirical predictions of protein conformation. Annu Rev Biochem. 1978;47:251–276. doi: 10.1146/annurev.bi.47.070178.001343. [DOI] [PubMed] [Google Scholar]
  5. Ehrmann M., Boyd D., Beckwith J. Genetic analysis of membrane protein topology by a sandwich gene fusion approach. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7574–7578. doi: 10.1073/pnas.87.19.7574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ellis J., Carlin A., Steffes C., Wu J., Liu J., Rosen B. P. Topological analysis of the lysine-specific permease of Escherichia coli. Microbiology. 1995 Aug;141(Pt 8):1927–1935. doi: 10.1099/13500872-141-8-1927. [DOI] [PubMed] [Google Scholar]
  7. Engelman D. M., Steitz T. A., Goldman A. Identifying nonpolar transbilayer helices in amino acid sequences of membrane proteins. Annu Rev Biophys Biophys Chem. 1986;15:321–353. doi: 10.1146/annurev.bb.15.060186.001541. [DOI] [PubMed] [Google Scholar]
  8. Glaser P., Kunst F., Arnaud M., Coudart M. P., Gonzales W., Hullo M. F., Ionescu M., Lubochinsky B., Marcelino L., Moszer I. Bacillus subtilis genome project: cloning and sequencing of the 97 kb region from 325 degrees to 333 degrees. Mol Microbiol. 1993 Oct;10(2):371–384. [PubMed] [Google Scholar]
  9. Harper E. T., Rose G. D. Helix stop signals in proteins and peptides: the capping box. Biochemistry. 1993 Aug 3;32(30):7605–7609. doi: 10.1021/bi00081a001. [DOI] [PubMed] [Google Scholar]
  10. Heijne G. The distribution of positively charged residues in bacterial inner membrane proteins correlates with the trans-membrane topology. EMBO J. 1986 Nov;5(11):3021–3027. doi: 10.1002/j.1460-2075.1986.tb04601.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Honoré N., Cole S. T. Nucleotide sequence of the aroP gene encoding the general aromatic amino acid transport protein of Escherichia coli K-12: homology with yeast transport proteins. Nucleic Acids Res. 1990 Feb 11;18(3):653–653. doi: 10.1093/nar/18.3.653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ito K., Akiyama Y. In vivo analysis of integration of membrane proteins in Escherichia coli. Mol Microbiol. 1991 Sep;5(9):2243–2253. doi: 10.1111/j.1365-2958.1991.tb02154.x. [DOI] [PubMed] [Google Scholar]
  13. Jennings M. P., Anderson J. K., Beacham I. R. Cloning and molecular analysis of the Salmonella enterica ansP gene, encoding an L-asparagine permease. Microbiology. 1995 Jan;141(Pt 1):141–146. doi: 10.1099/00221287-141-1-141. [DOI] [PubMed] [Google Scholar]
  14. Lacatena R. M., Cellini A., Scavizzi F., Tocchini-Valentini G. P. Topological analysis of the human beta 2-adrenergic receptor expressed in Escherichia coli. Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10521–10525. doi: 10.1073/pnas.91.22.10521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. MONOD J., COHEN-BAZIRE G., COHN M. Sur la biosynthèse de la beta-galactosidase (lactase) chez Escherichia coli; la spécificité de l'induction. Biochim Biophys Acta. 1951 Nov;7(4):585–599. doi: 10.1016/0006-3002(51)90072-8. [DOI] [PubMed] [Google Scholar]
  16. Manoil C. Analysis of membrane protein topology using alkaline phosphatase and beta-galactosidase gene fusions. Methods Cell Biol. 1991;34:61–75. doi: 10.1016/s0091-679x(08)61676-3. [DOI] [PubMed] [Google Scholar]
  17. Manoil C., Mekalanos J. J., Beckwith J. Alkaline phosphatase fusions: sensors of subcellular location. J Bacteriol. 1990 Feb;172(2):515–518. doi: 10.1128/jb.172.2.515-518.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Nilsson I., von Heijne G. Fine-tuning the topology of a polytopic membrane protein: role of positively and negatively charged amino acids. Cell. 1990 Sep 21;62(6):1135–1141. doi: 10.1016/0092-8674(90)90390-z. [DOI] [PubMed] [Google Scholar]
  20. Pi J., Wookey P. J., Pittard A. J. Cloning and sequencing of the pheP gene, which encodes the phenylalanine-specific transport system of Escherichia coli. J Bacteriol. 1991 Jun;173(12):3622–3629. doi: 10.1128/jb.173.12.3622-3629.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pi J., Wookey P. J., Pittard A. J. Site-directed mutagenesis reveals the importance of conserved charged residues for the transport activity of the PheP permease of Escherichia coli. J Bacteriol. 1993 Nov;175(22):7500–7504. doi: 10.1128/jb.175.22.7500-7504.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Reizer J., Finley K., Kakuda D., MacLeod C. L., Reizer A., Saier M. H., Jr Mammalian integral membrane receptors are homologous to facilitators and antiporters of yeast, fungi, and eubacteria. Protein Sci. 1993 Jan;2(1):20–30. doi: 10.1002/pro.5560020103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sarsero J. P., Pittard A. J. Membrane topology analysis of Escherichia coli K-12 Mtr permease by alkaline phosphatase and beta-galactosidase fusions. J Bacteriol. 1995 Jan;177(2):297–306. doi: 10.1128/jb.177.2.297-306.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sato S., Suzuki H., Widyastuti U., Hotta Y., Tabata S. Identification and characterization of genes induced during sexual differentiation in Schizosaccharomyces pombe. Curr Genet. 1994 Jul;26(1):31–37. doi: 10.1007/BF00326301. [DOI] [PubMed] [Google Scholar]
  25. Schmidt A., Hall M. N., Koller A. Two FK506 resistance-conferring genes in Saccharomyces cerevisiae, TAT1 and TAT2, encode amino acid permeases mediating tyrosine and tryptophan uptake. Mol Cell Biol. 1994 Oct;14(10):6597–6606. doi: 10.1128/mcb.14.10.6597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sychrova H., Chevallier M. R. Cloning and sequencing of the Saccharomyces cerevisiae gene LYP1 coding for a lysine-specific permease. Yeast. 1993 Jul;9(7):771–782. doi: 10.1002/yea.320090711. [DOI] [PubMed] [Google Scholar]
  27. Vandeyar M. A., Weiner M. P., Hutton C. J., Batt C. A. A simple and rapid method for the selection of oligodeoxynucleotide-directed mutants. Gene. 1988 May 15;65(1):129–133. doi: 10.1016/0378-1119(88)90425-8. [DOI] [PubMed] [Google Scholar]
  28. Wookey P. J., Pittard J., Forrest S. M., Davidson B. E. Cloning of the tyrP gene and further characterization of the tyrosine-specific transport system in Escherichia coli K-12. J Bacteriol. 1984 Oct;160(1):169–174. doi: 10.1128/jb.160.1.169-174.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]

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