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. 1990 Jul;87(13):4937–4941. doi: 10.1073/pnas.87.13.4937

lac permease of Escherichia coli: topology and sequence elements promoting membrane insertion.

J Calamia 1, C Manoil 1
PMCID: PMC54236  PMID: 2164211

Abstract

The membrane topology of Escherichia coli lac permease was analyzed using a set of 36 lac permease-alkaline phosphatase (lacY-phoA) gene fusions. The level of enzymatic activity of alkaline phosphatase fused to a cytoplasmic membrane protein appears to reflect whether the fusion junction site normally faces the cytoplasm or periplasm. The alkaline phosphatase activities of cells expressing the lacY-phoA fusions distinguish between models previously proposed for the topology of lac permease and favor one with 12 transmembrane segments. This model is fully compatible with the results of earlier biochemical and immunological studies. The properties of fusions with junctions spanning two of the transmembrane segments at 2- or 3-amino acid intervals indicate that approximately half of the residues of either segment (9-11 amino acids) suffices to promote alkaline phosphatase translocation across the membrane. The additional transmembrane segment amino acids that are not required for this membrane insertion process may normally be needed in unfused lac permease after insertion for stable association with the membrane.

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Selected References

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

  1. Akiyama Y., Ito K. Topology analysis of the SecY protein, an integral membrane protein involved in protein export in Escherichia coli. EMBO J. 1987 Nov;6(11):3465–3470. doi: 10.1002/j.1460-2075.1987.tb02670.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boyd D., Beckwith J. Positively charged amino acid residues can act as topogenic determinants in membrane proteins. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9446–9450. doi: 10.1073/pnas.86.23.9446. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boyd D., Manoil C., Beckwith J. Determinants of membrane protein topology. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8525–8529. doi: 10.1073/pnas.84.23.8525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. 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]
  7. Gutierrez C., Barondess J., Manoil C., Beckwith J. The use of transposon TnphoA to detect genes for cell envelope proteins subject to a common regulatory stimulus. Analysis of osmotically regulated genes in Escherichia coli. J Mol Biol. 1987 May 20;195(2):289–297. doi: 10.1016/0022-2836(87)90650-4. [DOI] [PubMed] [Google Scholar]
  8. Gött P., Boos W. The transmembrane topology of the sn-glycerol-3-phosphate permease of Escherichia coli analysed by phoA and lacZ protein fusions. Mol Microbiol. 1988 Sep;2(5):655–663. doi: 10.1111/j.1365-2958.1988.tb00074.x. [DOI] [PubMed] [Google Scholar]
  9. Ito K., Bassford P. J., Jr, Beckwith J. Protein localization in E. coli: is there a common step in the secretion of periplasmic and outer-membrane proteins? Cell. 1981 Jun;24(3):707–717. doi: 10.1016/0092-8674(81)90097-0. [DOI] [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. Kaback H. R. Use of site-directed mutagenesis to study the mechanism of a membrane transport protein. Biochemistry. 1987 Apr 21;26(8):2071–2076. doi: 10.1021/bi00382a001. [DOI] [PubMed] [Google Scholar]
  12. Levinson A., Silver D., Seed B. Minimal size plasmids containing an M13 origin for production of single-strand transducing particles. J Mol Appl Genet. 1984;2(6):507–517. [PubMed] [Google Scholar]
  13. Manoil C., Beckwith J. A genetic approach to analyzing membrane protein topology. Science. 1986 Sep 26;233(4771):1403–1408. doi: 10.1126/science.3529391. [DOI] [PubMed] [Google Scholar]
  14. Manoil C., Beckwith J. TnphoA: a transposon probe for protein export signals. Proc Natl Acad Sci U S A. 1985 Dec;82(23):8129–8133. doi: 10.1073/pnas.82.23.8129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Manoil C., Boyd D., Beckwith J. Molecular genetic analysis of membrane protein topology. Trends Genet. 1988 Aug;4(8):223–226. doi: 10.1016/0168-9525(88)90154-0. [DOI] [PubMed] [Google Scholar]
  16. Michaelis S., Inouye H., Oliver D., Beckwith J. Mutations that alter the signal sequence of alkaline phosphatase in Escherichia coli. J Bacteriol. 1983 Apr;154(1):366–374. doi: 10.1128/jb.154.1.366-374.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Page M. G., Rosenbusch J. P. Topography of lactose permease from Escherichia coli. J Biol Chem. 1988 Nov 5;263(31):15906–15914. [PubMed] [Google Scholar]
  18. Roepe P. D., Zbar R. I., Sarkar H. K., Kaback H. R. A five-residue sequence near the carboxyl terminus of the polytopic membrane protein lac permease is required for stability within the membrane. Proc Natl Acad Sci U S A. 1989 Jun;86(11):3992–3996. doi: 10.1073/pnas.86.11.3992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Spiess M., Handschin C. Deletion analysis of the internal signal-anchor domain of the human asialoglycoprotein receptor H1. EMBO J. 1987 Sep;6(9):2683–2691. doi: 10.1002/j.1460-2075.1987.tb02560.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Tabor S., Richardson C. C. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4767–4771. doi: 10.1073/pnas.84.14.4767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. 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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