Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1989 Oct;171(10):5536–5541. doi: 10.1128/jb.171.10.5536-5541.1989

Use of phoA fusions to study the topology of the Escherichia coli inner membrane protein leader peptidase.

J L San Millan 1, D Boyd 1, R Dalbey 1, W Wickner 1, J Beckwith 1
PMCID: PMC210394  PMID: 2551889

Abstract

A topology of the Escherichia coli leader peptidase has been previously proposed on the basis of proteolytic studies. Here, a collection of alkaline phosphatase fusions to leader peptidase is described. Fusions to the periplasmic domain of this protein exhibit high alkaline phosphatase activity, while fusions to the cytoplasmic domain exhibit low activity. Elements within the cytoplasmic domain are necessary to stably anchor alkaline phosphatase in the cytoplasm. The amino-terminal hydrophobic segment of leader peptidase acts as a weak export signal for alkaline phosphatase. However, when this segment is preceded by four lysines, it acts as a highly efficient export signal. The coherence of in vitro studies with alkaline phosphatase fusion analysis of the topology of leader peptidase further indicates the utility of this genetic approach to membrane protein structure and insertion.

Full text

PDF
5536

Images in this article

5539Image
on p.5539

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., 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]
  3. Chamberlain J. P. Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. Anal Biochem. 1979 Sep 15;98(1):132–135. doi: 10.1016/0003-2697(79)90716-4. [DOI] [PubMed] [Google Scholar]
  4. Chun S. Y., Parkinson J. S. Bacterial motility: membrane topology of the Escherichia coli MotB protein. Science. 1988 Jan 15;239(4837):276–278. doi: 10.1126/science.2447650. [DOI] [PubMed] [Google Scholar]
  5. Dalbey R. E., Wickner W. Leader peptidase catalyzes the release of exported proteins from the outer surface of the Escherichia coli plasma membrane. J Biol Chem. 1985 Dec 15;260(29):15925–15931. [PubMed] [Google Scholar]
  6. Eghtedarzadeh M. K., Henikoff S. Use of oligonucleotides to generate large deletions. Nucleic Acids Res. 1986 Jun 25;14(12):5115–5115. doi: 10.1093/nar/14.12.5115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Froshauer S., Green G. N., Boyd D., McGovern K., Beckwith J. Genetic analysis of the membrane insertion and topology of MalF, a cytoplasmic membrane protein of Escherichia coli. J Mol Biol. 1988 Apr 5;200(3):501–511. doi: 10.1016/0022-2836(88)90539-6. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. 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]
  11. Kieny M. P., Lathe R., Lecocq J. P. New versatile cloning and sequencing vectors based on bacteriophage M13. Gene. 1983 Dec;26(1):91–99. doi: 10.1016/0378-1119(83)90039-2. [DOI] [PubMed] [Google Scholar]
  12. Li P., Beckwith J., Inouye H. Alteration of the amino terminus of the mature sequence of a periplasmic protein can severely affect protein export in Escherichia coli. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7685–7689. doi: 10.1073/pnas.85.20.7685. [DOI] [PMC free article] [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. Moore K. E., Miura S. A small hydrophobic domain anchors leader peptidase to the cytoplasmic membrane of Escherichia coli. J Biol Chem. 1987 Jun 25;262(18):8806–8813. [PubMed] [Google Scholar]
  18. 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]
  19. Szczesna-Skorupa E., Browne N., Mead D., Kemper B. Positive charges at the NH2 terminus convert the membrane-anchor signal peptide of cytochrome P-450 to a secretory signal peptide. Proc Natl Acad Sci U S A. 1988 Feb;85(3):738–742. doi: 10.1073/pnas.85.3.738. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Wolfe P. B., Wickner W., Goodman J. M. Sequence of the leader peptidase gene of Escherichia coli and the orientation of leader peptidase in the bacterial envelope. J Biol Chem. 1983 Oct 10;258(19):12073–12080. [PubMed] [Google Scholar]
  21. von Heijne G., Wickner W., Dalbey R. E. The cytoplasmic domain of Escherichia coli leader peptidase is a "translocation poison" sequence. Proc Natl Acad Sci U S A. 1988 May;85(10):3363–3366. doi: 10.1073/pnas.85.10.3363. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES