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. 1994 Feb;176(3):563–568. doi: 10.1128/jb.176.3.563-568.1994

Selection of functional signal peptide cleavage sites from a library of random sequences.

T Palzkill 1, Q Q Le 1, A Wong 1, D Botstein 1
PMCID: PMC205091  PMID: 8300511

Abstract

The export of proteins to the periplasmic compartment of bacterial cells is mediated by an amino-terminal signal peptide. After transport, the signal peptide is cleaved by a processing enzyme, signal peptidase I. A comparison of the cleavage sites of many exported proteins has identified a conserved feature of small, uncharged amino acids at positions -1 and -3 relative to the cleavage site. To determine experimentally the sequences required for efficient signal peptide cleavage, we simultaneously randomized the amino acid residues from positions -4 to +2 of the TEM-1 beta-lactamase enzyme to form a library of random sequences. Mutants that provide wild-type levels of ampicillin resistance were then selected from the random-sequence library. The sequences of 15 mutants indicated a bias towards small amino acids. The N-terminal amino acid sequence of the mature enzyme was determined for nine of the mutants to assign the new -1 and -3 residues. Alanine was present in the -1 position for all nine of these mutants, strongly supporting the importance of alanine at the -1 position. The amino acids at the -3 position were much less conserved but were consistent with the -3 rules derived from sequence comparisons. Compared with the wild type, two of the nine mutants have an altered cleavage position, suggesting that sequence is more important than position for processing of the signal peptide.

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Selected References

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

  1. Barkocy-Gallagher G. A., Bassford P. J., Jr Synthesis of precursor maltose-binding protein with proline in the +1 position of the cleavage site interferes with the activity of Escherichia coli signal peptidase I in vivo. J Biol Chem. 1992 Jan 15;267(2):1231–1238. [PubMed] [Google Scholar]
  2. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  3. Cartwright S. J., Waley S. G. Purification of beta-lactamases by affinity chromatography on phenylboronic acid-agarose. Biochem J. 1984 Jul 15;221(2):505–512. doi: 10.1042/bj2210505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Datta N., Kontomichalou P. Penicillinase synthesis controlled by infectious R factors in Enterobacteriaceae. Nature. 1965 Oct 16;208(5007):239–241. doi: 10.1038/208239a0. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Fikes J. D., Barkocy-Gallagher G. A., Klapper D. G., Bassford P. J., Jr Maturation of Escherichia coli maltose-binding protein by signal peptidase I in vivo. Sequence requirements for efficient processing and demonstration of an alternate cleavage site. J Biol Chem. 1990 Feb 25;265(6):3417–3423. [PubMed] [Google Scholar]
  8. 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]
  9. Fitts R., Reuveny Z., van Amsterdam J., Mulholland J., Botstein D. Substitution of tyrosine for either cysteine in beta-lactamase prevents release from the membrane during secretion. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8540–8543. doi: 10.1073/pnas.84.23.8540. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Innis M. A., Tokunaga M., Williams M. E., Loranger J. M., Chang S. Y., Chang S., Wu H. C. Nucleotide sequence of the Escherichia coli prolipoprotein signal peptidase (lsp) gene. Proc Natl Acad Sci U S A. 1984 Jun;81(12):3708–3712. doi: 10.1073/pnas.81.12.3708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Laforet G. A., Kendall D. A. Functional limits of conformation, hydrophobicity, and steric constraints in prokaryotic signal peptide cleavage regions. Wild type transport by a simple polymeric signal sequence. J Biol Chem. 1991 Jan 15;266(2):1326–1334. [PubMed] [Google Scholar]
  13. 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]
  14. Mormeneo S., Knott R., Perlman D. Precise nucleotide sequence modifications with bidirectionally cleaving class-IIS excision linkers. Gene. 1987;61(1):21–30. doi: 10.1016/0378-1119(87)90361-1. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. O'Callaghan C. H., Morris A., Kirby S. M., Shingler A. H. Novel method for detection of beta-lactamases by using a chromogenic cephalosporin substrate. Antimicrob Agents Chemother. 1972 Apr;1(4):283–288. doi: 10.1128/aac.1.4.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Palzkill T., Botstein D. Identification of amino acid substitutions that alter the substrate specificity of TEM-1 beta-lactamase. J Bacteriol. 1992 Aug;174(16):5237–5243. doi: 10.1128/jb.174.16.5237-5243.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Palzkill T., Botstein D. Probing beta-lactamase structure and function using random replacement mutagenesis. Proteins. 1992 Sep;14(1):29–44. doi: 10.1002/prot.340140106. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Shackleton J. B., Robinson C. Transport of proteins into chloroplasts. The thylakoidal processing peptidase is a signal-type peptidase with stringent substrate requirements at the -3 and -1 positions. J Biol Chem. 1991 Jul 5;266(19):12152–12156. [PubMed] [Google Scholar]
  22. Shen L. M., Lee J. I., Cheng S. Y., Jutte H., Kuhn A., Dalbey R. E. Use of site-directed mutagenesis to define the limits of sequence variation tolerated for processing of the M13 procoat protein by the Escherichia coli leader peptidase. Biochemistry. 1991 Dec 24;30(51):11775–11781. doi: 10.1021/bi00115a006. [DOI] [PubMed] [Google Scholar]
  23. von Heijne G. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 1986 Jun 11;14(11):4683–4690. doi: 10.1093/nar/14.11.4683. [DOI] [PMC free article] [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. The signal peptide. J Membr Biol. 1990 May;115(3):195–201. doi: 10.1007/BF01868635. [DOI] [PubMed] [Google Scholar]

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