Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1995 Oct 24;92(22):10133–10136. doi: 10.1073/pnas.92.22.10133

High-affinity binding of Escherichia coli SecB to the signal sequence region of a presecretory protein.

M Watanabe 1, G Blobel 1
PMCID: PMC40750  PMID: 7479740

Abstract

The Escherichia coli cytosolic homotetrameric protein SecB is known to be involved in protein export across the plasma membrane. A currently prevalent view holds that SecB functions exclusively as a chaperone interacting nonspecifically with unfolded proteins, not necessarily exported proteins, whereas a contrary view holds that SecB functions primarily as a specific signal-recognition factor--i.e., in binding to the signal sequence region of exported proteins. To experimentally resolve these differences we assayed for binding between chemically pure SecB and chemically pure precursor (p) form (containing a signal sequence) and mature (m) form (lacking a signal sequence) of a model secretory protein (maltose binding protein, MBP) that was C-terminally truncated. Because of the C-terminal truncation, neither p nor m was able to fold. We found that SecB bound with 100-fold higher affinity to p (Kd 0.8 nM) than it bound to m (Kd 80 nM). As the presence of the signal sequence in p is the only feature that distinguished p from m, these data strongly suggest that the high-affinity binding of SecB is to the signal sequence region and not the mature region of p. Consistent with this conclusion, we found that a wild-type signal peptide, but not an export-incompetent mutant signal peptide of another exported protein (LamB), competed for binding to p. Moreover, the high-affinity binding of SecB to p was resistant to 1 M salt, whereas the low-affinity binding of SecB to m was not. These qualitative differences suggested that SecB binding to m was primarily by electrostatic interactions, whereas SecB binding to p was primarily via hydrophobic interactions, presumably with the hydrophobic core of the signal sequence. Taken together our data strongly support the notion that SecB is primarily a specific signal-recognition factor.

Full text

PDF
10133

Images in this article

10134Fig. 1
on p.10134
10135Fig. 2
on p.10135
10135Fig. 3
on p.10135

Selected References

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

  1. Collier D. N., Bankaitis V. A., Weiss J. B., Bassford P. J., Jr The antifolding activity of SecB promotes the export of the E. coli maltose-binding protein. Cell. 1988 Apr 22;53(2):273–283. doi: 10.1016/0092-8674(88)90389-3. [DOI] [PubMed] [Google Scholar]
  2. Crooke E., Guthrie B., Lecker S., Lill R., Wickner W. ProOmpA is stabilized for membrane translocation by either purified E. coli trigger factor or canine signal recognition particle. Cell. 1988 Sep 23;54(7):1003–1011. doi: 10.1016/0092-8674(88)90115-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Emr S. D., Silhavy T. J. Mutations affecting localization of an Escherichia coli outer membrane protein, the bacteriophage lambda receptor. J Mol Biol. 1980 Jul 25;141(1):63–90. doi: 10.1016/s0022-2836(80)80029-5. [DOI] [PubMed] [Google Scholar]
  4. Guthrie B., Wickner W. Trigger factor depletion or overproduction causes defective cell division but does not block protein export. J Bacteriol. 1990 Oct;172(10):5555–5562. doi: 10.1128/jb.172.10.5555-5562.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Kumamoto C. A. Escherichia coli SecB protein associates with exported protein precursors in vivo. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5320–5324. doi: 10.1073/pnas.86.14.5320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kumamoto C. A., Nault A. K. Characterization of the Escherichia coli protein-export gene secB. Gene. 1989 Jan 30;75(1):167–175. doi: 10.1016/0378-1119(89)90393-4. [DOI] [PubMed] [Google Scholar]
  8. McKnight C. J., Briggs M. S., Gierasch L. M. Functional and nonfunctional LamB signal sequences can be distinguished by their biophysical properties. J Biol Chem. 1989 Oct 15;264(29):17293–17297. [PubMed] [Google Scholar]
  9. Miller J. D., Wilhelm H., Gierasch L., Gilmore R., Walter P. GTP binding and hydrolysis by the signal recognition particle during initiation of protein translocation. Nature. 1993 Nov 25;366(6453):351–354. doi: 10.1038/366351a0. [DOI] [PubMed] [Google Scholar]
  10. Moore M. S., Blobel G. The two steps of nuclear import, targeting to the nuclear envelope and translocation through the nuclear pore, require different cytosolic factors. Cell. 1992 Jun 12;69(6):939–950. doi: 10.1016/0092-8674(92)90613-h. [DOI] [PubMed] [Google Scholar]
  11. Randall L. L., Topping T. B., Hardy S. J. No specific recognition of leader peptide by SecB, a chaperone involved in protein export. Science. 1990 May 18;248(4957):860–863. doi: 10.1126/science.2188362. [DOI] [PubMed] [Google Scholar]
  12. Simon S. M., Blobel G. A protein-conducting channel in the endoplasmic reticulum. Cell. 1991 May 3;65(3):371–380. doi: 10.1016/0092-8674(91)90455-8. [DOI] [PubMed] [Google Scholar]
  13. Simon S. M., Peskin C. S., Oster G. F. What drives the translocation of proteins? Proc Natl Acad Sci U S A. 1992 May 1;89(9):3770–3774. doi: 10.1073/pnas.89.9.3770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Watanabe M., Blobel G. Binding of a soluble factor of Escherichia coli to preproteins does not require ATP and appears to be the first step in protein export. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2248–2252. doi: 10.1073/pnas.86.7.2248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Watanabe M., Blobel G. Cytosolic factor purified from Escherichia coli is necessary and sufficient for the export of a preprotein and is a homotetramer of SecB. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2728–2732. doi: 10.1073/pnas.86.8.2728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Watanabe M., Blobel G. SecA protein is required for translocation of a model precursor protein into inverted vesicles of Escherichia coli plasma membrane. Proc Natl Acad Sci U S A. 1993 Oct 1;90(19):9011–9015. doi: 10.1073/pnas.90.19.9011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Watanabe M., Blobel G. SecB functions as a cytosolic signal recognition factor for protein export in E. coli. Cell. 1989 Aug 25;58(4):695–705. doi: 10.1016/0092-8674(89)90104-9. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES