Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1987 Jun;169(6):2345–2351. doi: 10.1128/jb.169.6.2345-2351.1987

Mutational alterations affecting the export competence of a truncated but fully functional maltose-binding protein signal peptide.

J D Fikes, V A Bankaitis, J P Ryan, P J Bassford Jr
PMCID: PMC212057  PMID: 3294786

Abstract

The wild-type maltose-binding protein (MBP) signal peptide is 26 amino acids in length. A mutationally altered MBP signal peptide has been previously described that is missing one of the basic residues from the hydrophilic segment and seven residues from the hydrophobic core; however, it still facilitates MBP secretion to the periplasm at a rate and efficiency comparable to those of the wild-type structure. Thus, this truncated signal peptide (designated the R2 signal peptide) must retain all of the essential features required for proper export function. In this study, alterations were obtained in the R2 signal peptide that resulted in an export-defective MBP. For the first time, signal sequence mutations were obtained that resulted in the synthesis of a totally export-defective MBP. As was previously the case for the wild-type signal peptide, the introduction of either charged residues or helix-breaking proline residues adversely affected export function. Despite these similarities, the position of these alterations within the R2 signal peptide, their relative effects on MBP secretion and processing, and an analysis of the ability of various extragenic prl mutations to suppress the secretion defects provide additional insight into the minimal requirements for a functional MBP signal peptide.

Full text

PDF
2345

Images in this article

2348Image
on p.2348
2348Image
on p.2348
2349Image
on p.2349

Selected References

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

  1. Bankaitis V. A., Bassford P. J., Jr Proper interaction between at least two components is required for efficient export of proteins to the Escherichia coli cell envelope. J Bacteriol. 1985 Jan;161(1):169–178. doi: 10.1128/jb.161.1.169-178.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bankaitis V. A., Bassford P. J., Jr The synthesis of export-defective proteins can interfere with normal protein export in Escherichia coli. J Biol Chem. 1984 Oct 10;259(19):12193–12200. [PubMed] [Google Scholar]
  3. Bankaitis V. A., Rasmussen B. A., Bassford P. J., Jr Intragenic suppressor mutations that restore export of maltose binding protein with a truncated signal peptide. Cell. 1984 May;37(1):243–252. doi: 10.1016/0092-8674(84)90320-9. [DOI] [PubMed] [Google Scholar]
  4. Bassford P. J., Jr, Silhavy T. J., Beckwith J. R. Use of gene fusion to study secretion of maltose-binding protein into Escherichia coli periplasm. J Bacteriol. 1979 Jul;139(1):19–31. doi: 10.1128/jb.139.1.19-31.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bassford P., Beckwith J. Escherichia coli mutants accumulating the precursor of a secreted protein in the cytoplasm. Nature. 1979 Feb 15;277(5697):538–541. doi: 10.1038/277538a0. [DOI] [PubMed] [Google Scholar]
  6. Bedouelle H., Bassford P. J., Jr, Fowler A. V., Zabin I., Beckwith J., Hofnung M. Mutations which alter the function of the signal sequence of the maltose binding protein of Escherichia coli. Nature. 1980 May 8;285(5760):78–81. doi: 10.1038/285078a0. [DOI] [PubMed] [Google Scholar]
  7. Casadaban M. J. Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J Mol Biol. 1976 Jul 5;104(3):541–555. doi: 10.1016/0022-2836(76)90119-4. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Débarbouillé M., Shuman H. A., Silhavy T. J., Schwartz M. Dominant constitutive mutations in malT, the positive regulator gene of the maltose regulon in Escherichia coli. J Mol Biol. 1978 Sep 15;124(2):359–371. doi: 10.1016/0022-2836(78)90304-2. [DOI] [PubMed] [Google Scholar]
  10. Emr S. D., Bassford P. J., Jr Localization and processing of outer membrane and periplasmic proteins in Escherichia coli strains harboring export-specific suppressor mutations. J Biol Chem. 1982 May 25;257(10):5852–5860. [PubMed] [Google Scholar]
  11. 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]
  12. Emr S. D., Silhavy T. J. Importance of secondary structure in the signal sequence for protein secretion. Proc Natl Acad Sci U S A. 1983 Aug;80(15):4599–4603. doi: 10.1073/pnas.80.15.4599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fikes J. D., Bassford P. J., Jr Export of unprocessed precursor maltose-binding protein to the periplasm of Escherichia coli cells. J Bacteriol. 1987 Jun;169(6):2352–2359. doi: 10.1128/jb.169.6.2352-2359.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Honig B. H., Hubbell W. L. Stability of "salt bridges" in membrane proteins. Proc Natl Acad Sci U S A. 1984 Sep;81(17):5412–5416. doi: 10.1073/pnas.81.17.5412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Neu H. C., Heppel L. A. The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. J Biol Chem. 1965 Sep;240(9):3685–3692. [PubMed] [Google Scholar]
  18. Perlman D., Halvorson H. O. A putative signal peptidase recognition site and sequence in eukaryotic and prokaryotic signal peptides. J Mol Biol. 1983 Jun 25;167(2):391–409. doi: 10.1016/s0022-2836(83)80341-6. [DOI] [PubMed] [Google Scholar]
  19. Rasmussen B. A., Bassford P. J., Jr Both linked and unlinked mutations can alter the intracellular site of synthesis of exported proteins of Escherichia coli. J Bacteriol. 1985 Jan;161(1):258–264. doi: 10.1128/jb.161.1.258-264.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Ryan J. P., Duncan M. C., Bankaitis V. A., Bassford P. J., Jr Intragenic reversion mutations that improve export of maltose-binding protein in Escherichia coli malE signal sequence mutants. J Biol Chem. 1986 Mar 5;261(7):3389–3395. [PubMed] [Google Scholar]
  22. Silhavy T. J., Benson S. A., Emr S. D. Mechanisms of protein localization. Microbiol Rev. 1983 Sep;47(3):313–344. doi: 10.1128/mr.47.3.313-344.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. von Heijne G. How signal sequences maintain cleavage specificity. J Mol Biol. 1984 Feb 25;173(2):243–251. doi: 10.1016/0022-2836(84)90192-x. [DOI] [PubMed] [Google Scholar]
  24. von Heijne G. Net N-C charge imbalance may be important for signal sequence function in bacteria. J Mol Biol. 1986 Nov 20;192(2):287–290. doi: 10.1016/0022-2836(86)90365-7. [DOI] [PubMed] [Google Scholar]
  25. von Heijne G. Patterns of amino acids near signal-sequence cleavage sites. Eur J Biochem. 1983 Jun 1;133(1):17–21. doi: 10.1111/j.1432-1033.1983.tb07424.x. [DOI] [PubMed] [Google Scholar]
  26. 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]

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

RESOURCES