Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1989 Sep;171(9):4609–4616. doi: 10.1128/jb.171.9.4609-4616.1989

Genetic studies on the inability of beta-galactosidase to be translocated across the Escherichia coli cytoplasmic membrane.

C Lee 1, P Li 1, H Inouye 1, E R Brickman 1, J Beckwith 1
PMCID: PMC210258  PMID: 2527843

Abstract

When a signal sequence is attached to beta-galactosidase, the normally cytoplasmic protein is unable to fully traverse the cytoplasmic membrane. We used a genetic approach to study those features of beta-galactosidase responsible for the block in translocation. By using both in vivo and in vitro techniques, fragments of beta-galactosidase were interposed between a signal sequence and alkaline phosphatase. The alkaline phosphatase acts as a sensor for any blocking effects of beta-galactosidase on export. From these studies, we show that multiple regions of beta-galactosidase contribute to its failure to be translocated. These results are most easily interpreted if the folding of beta-galactosidase or of domains of it is responsible for the block in export. In addition, in certain constructs, positively charged amino acids directly following the signal sequence interfered with export.

Full text

PDF
4609

Selected References

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

  1. 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]
  2. 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]
  3. 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]
  4. Beckwith J., Ferro-Novick S. Genetic studies on protein export in bacteria. Curr Top Microbiol Immunol. 1986;125:5–27. doi: 10.1007/978-3-642-71251-7_2. [DOI] [PubMed] [Google Scholar]
  5. Emr S. D., Schauer I., Hansen W., Esmon P., Schekman R. Invertase beta-galactosidase hybrid proteins fail to be transported from the endoplasmic reticulum in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Nov;4(11):2347–2355. doi: 10.1128/mcb.4.11.2347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gannon P. M., Li P., Kumamoto C. A. The mature portion of Escherichia coli maltose-binding protein (MBP) determines the dependence of MBP on SecB for export. J Bacteriol. 1989 Feb;171(2):813–818. doi: 10.1128/jb.171.2.813-818.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Kunkel T. A. The mutational specificity of DNA polymerase-beta during in vitro DNA synthesis. Production of frameshift, base substitution, and deletion mutations. J Biol Chem. 1985 May 10;260(9):5787–5796. [PubMed] [Google Scholar]
  10. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. 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]
  14. 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]
  15. Newton W. A., Beckwith J. R., Zipser D., Brenner S. Nonsense mutants and polarity in the lac operon of Escherichia coli. J Mol Biol. 1965 Nov;14(1):290–296. doi: 10.1016/s0022-2836(65)80250-9. [DOI] [PubMed] [Google Scholar]
  16. Randall L. L., Hardy S. J. Correlation of competence for export with lack of tertiary structure of the mature species: a study in vivo of maltose-binding protein in E. coli. Cell. 1986 Sep 12;46(6):921–928. doi: 10.1016/0092-8674(86)90074-7. [DOI] [PubMed] [Google Scholar]
  17. Rasmussen B. A., Bankaitis V. A., Bassford P. J., Jr Export and processing of MalE-LacZ hybrid proteins in Escherichia coli. J Bacteriol. 1984 Nov;160(2):612–617. doi: 10.1128/jb.160.2.612-617.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Sarthy A., Michaelis S., Beckwith J. Use of gene fusions to determine the orientation of gene phoA on the Escherichia coli chromosome. J Bacteriol. 1981 Jan;145(1):293–298. doi: 10.1128/jb.145.1.293-298.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Schwartz M., Roa M., Débarbouillé M. Mutations that affect lamB gene expression at a posttranscriptional level. Proc Natl Acad Sci U S A. 1981 May;78(5):2937–2941. doi: 10.1073/pnas.78.5.2937. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES