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. 1990 Feb;172(2):515–518. doi: 10.1128/jb.172.2.515-518.1990

Alkaline phosphatase fusions: sensors of subcellular location.

C Manoil 1, J J Mekalanos 1, J Beckwith 1
PMCID: PMC208471  PMID: 2404939

Abstract

Alkaline phosphatase fusions allow genes to be identified solely on the basis of their protein products being exported from the cytoplasm. Thus, the use of such fusions helps render biological processes which involve cell envelope and secreted proteins accessible to a sophisticated genetic analysis. Furthermore, alkaline phosphatase fusions can be used to locate export signals. Specifying such signals is an important component of studies on the structure of individual cell envelope proteins. The basis of the alkaline phosphatase fusion approach is the finding that the activity of the enzyme responds differently to different environments. Thus, the activity of the fusion protein gives evidence as to its location. This general approach of using sensor proteins which vary in their function, depending on their environment, could be extended to the study of other sorts of problems. It may be that certain enzymes will provide an assay for localization to a particular subcellular compartment, if the environment of the compartment differs from that of others. For instance, the lysosome is more acidic than other intracellular organelles. A gene fusion system employing a reporter enzyme that could show activity only at the pH of the lysosome could allow the detection of signals determining lysosomal localization. Analogous types of enzymes may be used as probes for other subcellular compartments.

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Selected References

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

  1. Ahmad M., Bussey H. Topology of membrane insertion in vitro and plasma membrane assembly in vivo of the yeast arginine permease. Mol Microbiol. 1988 Sep;2(5):627–635. doi: 10.1111/j.1365-2958.1988.tb00071.x. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Boquet P. L., Manoil C., Beckwith J. Use of TnphoA to detect genes for exported proteins in Escherichia coli: identification of the plasmid-encoded gene for a periplasmic acid phosphatase. J Bacteriol. 1987 Apr;169(4):1663–1669. doi: 10.1128/jb.169.4.1663-1669.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Broome-Smith J. K., Spratt B. G. A vector for the construction of translational fusions to TEM beta-lactamase and the analysis of protein export signals and membrane protein topology. Gene. 1986;49(3):341–349. doi: 10.1016/0378-1119(86)90370-7. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Coulton J. W., Reid G. K., Campana A. Export of hybrid proteins FhuA'-'LacZ and FhuA'-'PhoA to the cell envelope of Escherichia coli K-12. J Bacteriol. 1988 May;170(5):2267–2275. doi: 10.1128/jb.170.5.2267-2275.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Edelman A., Bowler L., Broome-Smith J. K., Spratt B. G. Use of a beta-lactamase fusion vector to investigate the organization of penicillin-binding protein 1B in the cytoplasmic membrane of Escherichia coli. Mol Microbiol. 1987 Jul;1(1):101–106. doi: 10.1111/j.1365-2958.1987.tb00533.x. [DOI] [PubMed] [Google Scholar]
  9. Finlay B. B., Starnbach M. N., Francis C. L., Stocker B. A., Chatfield S., Dougan G., Falkow S. Identification and characterization of TnphoA mutants of Salmonella that are unable to pass through a polarized MDCK epithelial cell monolayer. Mol Microbiol. 1988 Nov;2(6):757–766. doi: 10.1111/j.1365-2958.1988.tb00087.x. [DOI] [PubMed] [Google Scholar]
  10. Forst S., Comeau D., Norioka S., Inouye M. Localization and membrane topology of EnvZ, a protein involved in osmoregulation of OmpF and OmpC in Escherichia coli. J Biol Chem. 1987 Dec 5;262(34):16433–16438. [PubMed] [Google Scholar]
  11. 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]
  12. Georgiou C. D., Dueweke T. J., Gennis R. B. Beta-galactosidase gene fusions as probes for the cytoplasmic regions of subunits I and II of the membrane-bound cytochrome d terminal oxidase from Escherichia coli. J Biol Chem. 1988 Sep 15;263(26):13130–13137. [PubMed] [Google Scholar]
  13. Gething M. J., McCammon K., Sambrook J. Expression of wild-type and mutant forms of influenza hemagglutinin: the role of folding in intracellular transport. Cell. 1986 Sep 12;46(6):939–950. doi: 10.1016/0092-8674(86)90076-0. [DOI] [PubMed] [Google Scholar]
  14. Green G. N., Hansen W., Walter P. The use of gene-fusions to determine membrane protein topology in Saccharomyces cerevisiae. J Cell Sci Suppl. 1989;11:109–113. doi: 10.1242/jcs.1989.supplement_11.9. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Gutierrez C., Devedjian J. C. A plasmid facilitating in vitro construction of phoA gene fusions in Escherichia coli. Nucleic Acids Res. 1989 May 25;17(10):3999–3999. doi: 10.1093/nar/17.10.3999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Herrero M., de Lorenzo V., Neilands J. B. Nucleotide sequence of the iucD gene of the pColV-K30 aerobactin operon and topology of its product studied with phoA and lacZ gene fusions. J Bacteriol. 1988 Jan;170(1):56–64. doi: 10.1128/jb.170.1.56-64.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hinton J. C., Salmond G. P. Use of TnphoA to enrich for extracellular enzyme mutants of Erwinia carotovora subspecies carotovora. Mol Microbiol. 1987 Nov;1(3):381–386. doi: 10.1111/j.1365-2958.1987.tb01946.x. [DOI] [PubMed] [Google Scholar]
  20. Hoffman C. S., Wright A. Fusions of secreted proteins to alkaline phosphatase: an approach for studying protein secretion. Proc Natl Acad Sci U S A. 1985 Aug;82(15):5107–5111. doi: 10.1073/pnas.82.15.5107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Horabin J. I., Webster R. E. An amino acid sequence which directs membrane insertion causes loss of membrane potential. J Biol Chem. 1988 Aug 15;263(23):11575–11583. [PubMed] [Google Scholar]
  22. Knapp S., Mekalanos J. J. Two trans-acting regulatory genes (vir and mod) control antigenic modulation in Bordetella pertussis. J Bacteriol. 1988 Nov;170(11):5059–5066. doi: 10.1128/jb.170.11.5059-5066.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Long S., McCune S., Walker G. C. Symbiotic loci of Rhizobium meliloti identified by random TnphoA mutagenesis. J Bacteriol. 1988 Sep;170(9):4257–4265. doi: 10.1128/jb.170.9.4257-4265.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. 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]
  28. Marullo S., Delavier-Klutchko C., Eshdat Y., Strosberg A. D., Emorine L. Human beta 2-adrenergic receptors expressed in Escherichia coli membranes retain their pharmacological properties. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7551–7555. doi: 10.1073/pnas.85.20.7551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Michaelis S., Hunt J. F., Beckwith J. Effects of signal sequence mutations on the kinetics of alkaline phosphatase export to the periplasm in Escherichia coli. J Bacteriol. 1986 Jul;167(1):160–167. doi: 10.1128/jb.167.1.160-167.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Miller S. I., Kukral A. M., Mekalanos J. J. A two-component regulatory system (phoP phoQ) controls Salmonella typhimurium virulence. Proc Natl Acad Sci U S A. 1989 Jul;86(13):5054–5058. doi: 10.1073/pnas.86.13.5054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Miller V. L., Taylor R. K., Mekalanos J. J. Cholera toxin transcriptional activator toxR is a transmembrane DNA binding protein. Cell. 1987 Jan 30;48(2):271–279. doi: 10.1016/0092-8674(87)90430-2. [DOI] [PubMed] [Google Scholar]
  33. Peterson K. M., Mekalanos J. J. Characterization of the Vibrio cholerae ToxR regulon: identification of novel genes involved in intestinal colonization. Infect Immun. 1988 Nov;56(11):2822–2829. doi: 10.1128/iai.56.11.2822-2829.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Pradel E., Boquet P. L. Acid phosphatases of Escherichia coli: molecular cloning and analysis of agp, the structural gene for a periplasmic acid glucose phosphatase. J Bacteriol. 1988 Oct;170(10):4916–4923. doi: 10.1128/jb.170.10.4916-4923.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. San Millan J. L., Boyd D., Dalbey R., Wickner W., Beckwith J. Use of phoA fusions to study the topology of the Escherichia coli inner membrane protein leader peptidase. J Bacteriol. 1989 Oct;171(10):5536–5541. doi: 10.1128/jb.171.10.5536-5541.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sarkar H. K., Thorens B., Lodish H. F., Kaback H. R. Expression of the human erythrocyte glucose transporter in Escherichia coli. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5463–5467. doi: 10.1073/pnas.85.15.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sarthy A., Michaelis S., Beckwith J. Deletion map of the Escherichia coli structural gene for alkaline phosphatase, phoA. J Bacteriol. 1981 Jan;145(1):288–292. doi: 10.1128/jb.145.1.288-292.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sengstag C., Stirling C., Schekman R., Rine J. Genetic and biochemical evaluation of eucaryotic membrane protein topology: multiple transmembrane domains of Saccharomyces cerevisiae 3-hydroxy-3-methylglutaryl coenzyme A reductase. Mol Cell Biol. 1990 Feb;10(2):672–680. doi: 10.1128/mcb.10.2.672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Silhavy T. J., Beckwith J. R. Uses of lac fusions for the study of biological problems. Microbiol Rev. 1985 Dec;49(4):398–418. doi: 10.1128/mr.49.4.398-418.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Strauch K. L., Beckwith J. An Escherichia coli mutation preventing degradation of abnormal periplasmic proteins. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1576–1580. doi: 10.1073/pnas.85.5.1576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Strauch K. L., Johnson K., Beckwith J. Characterization of degP, a gene required for proteolysis in the cell envelope and essential for growth of Escherichia coli at high temperature. J Bacteriol. 1989 May;171(5):2689–2696. doi: 10.1128/jb.171.5.2689-2696.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Strom M. S., Lory S. Mapping of export signals of Pseudomonas aeruginosa pilin with alkaline phosphatase fusions. J Bacteriol. 1987 Jul;169(7):3181–3188. doi: 10.1128/jb.169.7.3181-3188.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Taylor R. K., Manoil C., Mekalanos J. J. Broad-host-range vectors for delivery of TnphoA: use in genetic analysis of secreted virulence determinants of Vibrio cholerae. J Bacteriol. 1989 Apr;171(4):1870–1878. doi: 10.1128/jb.171.4.1870-1878.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Taylor R. K., Miller V. L., Furlong D. B., Mekalanos J. J. Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin. Proc Natl Acad Sci U S A. 1987 May;84(9):2833–2837. doi: 10.1073/pnas.84.9.2833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Weber R. F., Silverman P. M. The cpx proteins of Escherichia coli K12. Structure of the cpxA polypeptide as an inner membrane component. J Mol Biol. 1988 Sep 20;203(2):467–478. doi: 10.1016/0022-2836(88)90013-7. [DOI] [PubMed] [Google Scholar]
  46. Winans S. C., Kerstetter R. A., Ward J. E., Nester E. W. A protein required for transcriptional regulation of Agrobacterium virulence genes spans the cytoplasmic membrane. J Bacteriol. 1989 Mar;171(3):1616–1622. doi: 10.1128/jb.171.3.1616-1622.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. d'Enfert C., Pugsley A. P. A gene fusion approach to the study of pullulanase export and secretion in Escherichia coli. Mol Microbiol. 1987 Sep;1(2):159–168. doi: 10.1111/j.1365-2958.1987.tb00508.x. [DOI] [PubMed] [Google Scholar]

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