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. 1986 Jul;167(1):160–167. doi: 10.1128/jb.167.1.160-167.1986

Effects of signal sequence mutations on the kinetics of alkaline phosphatase export to the periplasm in Escherichia coli.

S Michaelis, J F Hunt, J Beckwith
PMCID: PMC212855  PMID: 3522543

Abstract

We isolated a collection of mutants defective in the export of alkaline phosphatase to the periplasm. Two classes of mutants were obtained: one class with lesions unlinked to the phoA gene and a second class harboring linked mutations. Among the former class, one mutant is cold sensitive for growth and may be defective in a component of the Escherichia coli secretory apparatus. Included in the latter class are 47 mutants which are characterized in detail in this report. To facilitate DNA sequence analysis of these mutants, we devised a convenient method that relies on homologous recombination in vivo to transfer phoA mutations from the bacterial chromosome directly onto the genome of a single-stranded M13 phage vector. DNA sequence analysis revealed that our collection of mutants comprises six unique mutations, all of which reside in the phoA signal sequence coding region and lend further support to the notion that the length of the hydrophobic core of the signal sequence is crucial for its function in protein export. Kinetic studies showed that in these mutants, the small fraction of alkaline phosphatase which succeeds in reaching a periplasmic location, despite a defective signal sequence, is translocated across the membrane in a slow, posttranslational fashion.

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Selected References

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  1. Beckwith J., Silhavy T. J. Genetic analysis of protein export in Escherichia coli. Methods Enzymol. 1983;97:3–11. doi: 10.1016/0076-6879(83)97114-8. [DOI] [PubMed] [Google Scholar]
  2. Bedouelle H., Hofnung M. Functional implications of secondary structure analysis of wild type and mutant bacterial signal peptides. Prog Clin Biol Res. 1981;63:399–403. [PubMed] [Google Scholar]
  3. Benson S. A., Hall M. N., Silhavy T. J. Genetic analysis of protein export in Escherichia coli K12. Annu Rev Biochem. 1985;54:101–134. doi: 10.1146/annurev.bi.54.070185.000533. [DOI] [PubMed] [Google Scholar]
  4. Blobel G., Dobberstein B. Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J Cell Biol. 1975 Dec;67(3):835–851. doi: 10.1083/jcb.67.3.835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blobel G., Dobberstein B. Transfer of proteins across membranes. II. Reconstitution of functional rough microsomes from heterologous components. J Cell Biol. 1975 Dec;67(3):852–862. doi: 10.1083/jcb.67.3.852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bradshaw R. A., Cancedda F., Ericsson L. H., Neumann P. A., Piccoli S. P., Schlesinger M. J., Shriefer K., Walsh K. A. Amino acid sequence of Escherichia coli alkaline phosphatase. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3473–3477. doi: 10.1073/pnas.78.6.3473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Chen L., Rhoads D., Tai P. C. Alkaline phosphatase and OmpA protein can be translocated posttranslationally into membrane vesicles of Escherichia coli. J Bacteriol. 1985 Mar;161(3):973–980. doi: 10.1128/jb.161.3.973-980.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Date T., Wickner W. T. Procoat, the precursor of M13 coat protein, inserts post-translationally into the membrane of cells infected by wild-type virus. J Virol. 1981 Mar;37(3):1087–1089. doi: 10.1128/jvi.37.3.1087-1089.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Emr S. D., Hanley-Way S., Silhavy T. J. Suppressor mutations that restore export of a protein with a defective signal sequence. Cell. 1981 Jan;23(1):79–88. doi: 10.1016/0092-8674(81)90272-5. [DOI] [PubMed] [Google Scholar]
  11. Emr S. D., Silhavy T. J. Molecular components of the signal sequence that function in the initiation of protein export. J Cell Biol. 1982 Dec;95(3):689–696. doi: 10.1083/jcb.95.3.689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Garwin J. L., Beckwith J. Secretion and processing of ribose-binding protein in Escherichia coli. J Bacteriol. 1982 Feb;149(2):789–792. doi: 10.1128/jb.149.2.789-792.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hall M. N., Gabay J., Schwartz M. Evidence for a coupling of synthesis and export of an outer membrane protein in Escherichia coli. EMBO J. 1983;2(1):15–19. doi: 10.1002/j.1460-2075.1983.tb01373.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hall M. N., Schwartz M., Silhavy T. J. Sequence information within the lamB genes in required for proper routing of the bacteriophage lambda receptor protein to the outer membrane of Escherichia coli K-12. J Mol Biol. 1982 Mar 25;156(1):93–112. doi: 10.1016/0022-2836(82)90461-2. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Inouye H., Barnes W., Beckwith J. Signal sequence of alkaline phosphatase of Escherichia coli. J Bacteriol. 1982 Feb;149(2):434–439. doi: 10.1128/jb.149.2.434-439.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Inouye H., Michaelis S., Wright A., Beckwith J. Cloning and restriction mapping of the alkaline phosphatase structural gene (phoA) of Escherichia coli and generation of deletion mutants in vitro. J Bacteriol. 1981 May;146(2):668–675. doi: 10.1128/jb.146.2.668-675.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Inouye M., Halegoua S. Secretion and membrane localization of proteins in Escherichia coli. CRC Crit Rev Biochem. 1980;7(4):339–371. doi: 10.3109/10409238009105465. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Josefsson L. G., Randall L. L. Processing in vivo of precursor maltose-binding protein in Escherichia coli occurs post-translationally as well as co-translationally. J Biol Chem. 1981 Mar 10;256(5):2504–2507. [PubMed] [Google Scholar]
  21. Koshland D., Botstein D. Evidence for posttranslational translocation of beta-lactamase across the bacterial inner membrane. Cell. 1982 Oct;30(3):893–902. doi: 10.1016/0092-8674(82)90294-x. [DOI] [PubMed] [Google Scholar]
  22. Koshland D., Sauer R. T., Botstein D. Diverse effects of mutations in the signal sequence on the secretion of beta-lactamase in Salmonella typhimurium. Cell. 1982 Oct;30(3):903–914. doi: 10.1016/0092-8674(82)90295-1. [DOI] [PubMed] [Google Scholar]
  23. Kumamoto C. A., Beckwith J. Evidence for specificity at an early step in protein export in Escherichia coli. J Bacteriol. 1985 Jul;163(1):267–274. doi: 10.1128/jb.163.1.267-274.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kumamoto C. A., Oliver D. B., Beckwith J. Signal sequence mutations disrupt feedback between secretion of an exported protein and its synthesis in E. coli. 1984 Apr 26-May 2Nature. 308(5962):863–864. doi: 10.1038/308863a0. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Michaelis S., Beckwith J. Mechanism of incorporation of cell envelope proteins in Escherichia coli. Annu Rev Microbiol. 1982;36:435–465. doi: 10.1146/annurev.mi.36.100182.002251. [DOI] [PubMed] [Google Scholar]
  27. Michaelis S., Guarente L., Beckwith J. In vitro construction and characterization of phoA-lacZ gene fusions in Escherichia coli. J Bacteriol. 1983 Apr;154(1):356–365. doi: 10.1128/jb.154.1.356-365.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Müller M., Blobel G. In vitro translocation of bacterial proteins across the plasma membrane of Escherichia coli. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7421–7425. doi: 10.1073/pnas.81.23.7421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Oliver D. B., Beckwith J. E. coli mutant pleiotropically defective in the export of secreted proteins. Cell. 1981 Sep;25(3):765–772. doi: 10.1016/0092-8674(81)90184-7. [DOI] [PubMed] [Google Scholar]
  31. Randall L. L. Translocation of domains of nascent periplasmic proteins across the cytoplasmic membrane is independent of elongation. Cell. 1983 May;33(1):231–240. doi: 10.1016/0092-8674(83)90352-5. [DOI] [PubMed] [Google Scholar]
  32. Ryan J. P., Bassford P. J., Jr Post-translational export of maltose-binding protein in Escherichia coli strains harboring malE signal sequence mutations and either prl+ or prl suppressor alleles. J Biol Chem. 1985 Nov 25;260(27):14832–14837. [PubMed] [Google Scholar]
  33. Sarthy A., Fowler A., Zabin I., Beckwith J. Use of gene fusions to determine a partial signal sequence of alkaline phosphatase. J Bacteriol. 1979 Sep;139(3):932–939. doi: 10.1128/jb.139.3.932-939.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schlesinger M. J., Reynolds J. A., Schlesinger S. Formation and localization of the alkaline phosphatase of Escherichia coli. Ann N Y Acad Sci. 1969 Oct 14;166(2):368–379. doi: 10.1111/j.1749-6632.1969.tb46408.x. [DOI] [PubMed] [Google Scholar]
  35. Vlasuk G. P., Inouye S., Ito H., Itakura K., Inouye M. Effects of the complete removal of basic amino acid residues from the signal peptide on secretion of lipoprotein in Escherichia coli. J Biol Chem. 1983 Jun 10;258(11):7141–7148. [PubMed] [Google Scholar]
  36. Zimmermann R., Watts C., Wickner W. The biosynthesis of membrane-bound M13 coat protein. Energetics and assembly intermediates. J Biol Chem. 1982 Jun 10;257(11):6529–6536. [PubMed] [Google Scholar]

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