Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1994 Sep;176(18):5796–5801. doi: 10.1128/jb.176.18.5796-5801.1994

Effect of alteration of charged residues at the N termini of signal peptides on protein export in Bacillus subtilis.

M Chen 1, V Nagarajan 1
PMCID: PMC196784  PMID: 8083171

Abstract

The role of positively charged residues at the N termini of signal peptides in protein export has been studied in Bacillus subtilis. Bacillus signal peptides (alkaline protease [Apr] and neutral protease [Npr] from Bacillus amyloliquefaciens) were altered and fused to mature levansucrase (Lvs). The effects of the various alterations on the export of Lvs in B. subtilis were determined. The replacement of positively charged residues with neutral residues in both Apr and Npr signal peptides resulted in a slight defect in the export of Lvs from B. subtilis. Introduction of a negatively charged residue (aspartic acid) at the N terminus of Npr signal peptide blocked the export of Lvs. However, Apr signal peptide with a net charge of -3 (three aspartic acid residues) was still functional.

Full text

PDF
5798

Images in this article

5798Image
on p.5798
5799Image
on p.5799
5799Image
on p.5799
5799Image
on p.5799
5800Image
on p.5800

Selected References

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

  1. Akita M., Sasaki S., Matsuyama S., Mizushima S. SecA interacts with secretory proteins by recognizing the positive charge at the amino terminus of the signal peptide in Escherichia coli. J Biol Chem. 1990 May 15;265(14):8164–8169. [PubMed] [Google Scholar]
  2. Bieker-Brady K., Silhavy T. J. Suppressor analysis suggests a multistep, cyclic mechanism for protein secretion in Escherichia coli. EMBO J. 1992 Sep;11(9):3165–3174. doi: 10.1002/j.1460-2075.1992.tb05393.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Borchert T. V., Nagarajan V. Effect of signal sequence alterations on export of levansucrase in Bacillus subtilis. J Bacteriol. 1991 Jan;173(1):276–282. doi: 10.1128/jb.173.1.276-282.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chen M., Nagarajan V. The roles of signal peptide and mature protein in RNase (barnase) export from Bacillus subtilis. Mol Gen Genet. 1993 Jun;239(3):409–415. doi: 10.1007/BF00276939. [DOI] [PubMed] [Google Scholar]
  5. Gierasch L. M. Signal sequences. Biochemistry. 1989 Feb 7;28(3):923–930. doi: 10.1021/bi00429a001. [DOI] [PubMed] [Google Scholar]
  6. Hikita C., Mizushima S. Effects of total hydrophobicity and length of the hydrophobic domain of a signal peptide on in vitro translocation efficiency. J Biol Chem. 1992 Mar 5;267(7):4882–4888. [PubMed] [Google Scholar]
  7. Iino T., Takahashi M., Sako T. Role of amino-terminal positive charge on signal peptide in staphylokinase export across the cytoplasmic membrane of Escherichia coli. J Biol Chem. 1987 May 25;262(15):7412–7417. [PubMed] [Google Scholar]
  8. Inouye S., Soberon X., Franceschini T., Nakamura K., Itakura K., Inouye M. Role of positive charge on the amino-terminal region of the signal peptide in protein secretion across the membrane. Proc Natl Acad Sci U S A. 1982 Jun;79(11):3438–3441. doi: 10.1073/pnas.79.11.3438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kendall D. A., Bock S. C., Kaiser E. T. Idealization of the hydrophobic segment of the alkaline phosphatase signal peptide. Nature. 1986 Jun 12;321(6071):706–708. doi: 10.1038/321706a0. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Nagarajan V., Albertson H., Chen M., Ribbe J. Modular expression and secretion vectors for Bacillus subtilis. Gene. 1992 May 1;114(1):121–126. doi: 10.1016/0378-1119(92)90717-4. [DOI] [PubMed] [Google Scholar]
  12. Nagarajan V. System for secretion of heterologous proteins in Bacillus subtilis. Methods Enzymol. 1990;185:214–223. doi: 10.1016/0076-6879(90)85021-f. [DOI] [PubMed] [Google Scholar]
  13. Oliver D. B. SecA protein: autoregulated ATPase catalysing preprotein insertion and translocation across the Escherichia coli inner membrane. Mol Microbiol. 1993 Jan;7(2):159–165. doi: 10.1111/j.1365-2958.1993.tb01107.x. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Phoenix D. A., Kusters R., Hikita C., Mizushima S., de Kruijff B. OmpF-Lpp signal sequence mutants with varying charge hydrophobicity ratios provide evidence for a phosphatidylglycerol-signal sequence interaction during protein translocation across the Escherichia coli inner membrane. J Biol Chem. 1993 Aug 15;268(23):17069–17073. [PubMed] [Google Scholar]
  16. Pugsley A. P. The complete general secretory pathway in gram-negative bacteria. Microbiol Rev. 1993 Mar;57(1):50–108. doi: 10.1128/mr.57.1.50-108.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Puziss J. W., Fikes J. D., Bassford P. J., Jr Analysis of mutational alterations in the hydrophilic segment of the maltose-binding protein signal peptide. J Bacteriol. 1989 May;171(5):2303–2311. doi: 10.1128/jb.171.5.2303-2311.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Puziss J. W., Harvey R. J., Bassford P. J., Jr Alterations in the hydrophilic segment of the maltose-binding protein (MBP) signal peptide that affect either export or translation of MBP. J Bacteriol. 1992 Oct;174(20):6488–6497. doi: 10.1128/jb.174.20.6488-6497.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Puziss J. W., Strobel S. M., Bassford P. J., Jr Export of maltose-binding protein species with altered charge distribution surrounding the signal peptide hydrophobic core in Escherichia coli cells harboring prl suppressor mutations. J Bacteriol. 1992 Jan;174(1):92–101. doi: 10.1128/jb.174.1.92-101.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Schatz P. J., Beckwith J. Genetic analysis of protein export in Escherichia coli. Annu Rev Genet. 1990;24:215–248. doi: 10.1146/annurev.ge.24.120190.001243. [DOI] [PubMed] [Google Scholar]
  21. Stader J., Benson S. A., Silhavy T. J. Kinetic analysis of lamB mutants suggests the signal sequence plays multiple roles in protein export. J Biol Chem. 1986 Nov 15;261(32):15075–15080. [PubMed] [Google Scholar]
  22. Sung C. Y., Gennity J. M., Pollitt N. S., Inouye M. A positive residue in the hydrophobic core of the Escherichia coli lipoprotein signal peptide suppresses the secretion defect caused by an acidic amino terminus. J Biol Chem. 1992 Jan 15;267(2):997–1000. [PubMed] [Google Scholar]
  23. Thom J. R., Randall L. L. Role of the leader peptide of maltose-binding protein in two steps of the export process. J Bacteriol. 1988 Dec;170(12):5654–5661. doi: 10.1128/jb.170.12.5654-5661.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Vasantha N., Thompson L. D., Rhodes C., Banner C., Nagle J., Filpula D. Genes for alkaline protease and neutral protease from Bacillus amyloliquefaciens contain a large open reading frame between the regions coding for signal sequence and mature protein. J Bacteriol. 1984 Sep;159(3):811–819. doi: 10.1128/jb.159.3.811-819.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wickner W., Driessen A. J., Hartl F. U. The enzymology of protein translocation across the Escherichia coli plasma membrane. Annu Rev Biochem. 1991;60:101–124. doi: 10.1146/annurev.bi.60.070191.000533. [DOI] [PubMed] [Google Scholar]
  26. Yamane K., Akiyama Y., Ito K., Mizushima S. A positively charged region is a determinant of the orientation of cytoplasmic membrane proteins in Escherichia coli. J Biol Chem. 1990 Dec 5;265(34):21166–21171. [PubMed] [Google Scholar]
  27. van der Wolk J., Klose M., Breukink E., Demel R. A., de Kruijff B., Freudl R., Driessen A. J. Characterization of a Bacillus subtilis SecA mutant protein deficient in translocation ATPase and release from the membrane. Mol Microbiol. 1993 Apr;8(1):31–42. doi: 10.1111/j.1365-2958.1993.tb01200.x. [DOI] [PubMed] [Google Scholar]
  28. von Heijne G., Abrahmsén L. Species-specific variation in signal peptide design. Implications for protein secretion in foreign hosts. FEBS Lett. 1989 Feb 27;244(2):439–446. doi: 10.1016/0014-5793(89)80579-4. [DOI] [PubMed] [Google Scholar]

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

RESOURCES