• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of embojLink to Publisher's site
EMBO J. Nov 16, 1998; 17(22): 6487–6496.
PMCID: PMC1170996

Substrate-induced assembly of a contiguous channel for protein export from E.coli: reversible bridging of an inner-membrane translocase to an outer membrane exit pore.


The toxin HlyA is exported from Escherichia coli, without a periplasmic intermediate, by a type I system comprising an energized inner-membrane (IM) translocase of two proteins, HlyD and the traffic ATPase HlyB, and the outer-membrane (OM) porin-like TolC. These and the toxin substrate were expressed separately to reconstitute export and, via affinity tags on the IM proteins, cross-linked in vivo complexes were isolated before and after substrate engagement. HlyD and HlyB assembled a stable IM complex in the absence of TolC and substrate. Both engaged HlyA, inducing the IM complex to contact TolC, concomitant with conformational change in all three exporter components. The IM-OM bridge was formed primarily by HlyD, which assembled to stable IM trimers, corresponding to the OM trimers of TolC. The bridge was transient, components reverting to IM and OM states after translocation. Mutant HlyB that bound, but did not hydrolyse ATP, supported IM complex assembly, substrate recruitment and bridging, but HlyA stalled in the channel. A similar picture was evident when the HlyD C-terminus was masked. Export thus occurs via a contiguous channel which is formed, without traffic ATPase ATP hydrolysis, by substrate-induced, reversible bridging of the IM translocase to the OM export pore.

Full Text

The Full Text of this article is available as a PDF (414K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Dinh T, Paulsen IT, Saier MH., Jr A family of extracytoplasmic proteins that allow transport of large molecules across the outer membranes of gram-negative bacteria. J Bacteriol. 1994 Jul;176(13):3825–3831. [PMC free article] [PubMed]
  • Duong F, Eichler J, Price A, Leonard MR, Wickner W. Biogenesis of the gram-negative bacterial envelope. Cell. 1997 Nov 28;91(5):567–573. [PubMed]
  • Felmlee T, Welch RA. Alterations of amino acid repeats in the Escherichia coli hemolysin affect cytolytic activity and secretion. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5269–5273. [PMC free article] [PubMed]
  • Gadsby DC, Nairn AC. Regulation of CFTR channel gating. Trends Biochem Sci. 1994 Nov;19(11):513–518. [PubMed]
  • Gentschev I, Goebel W. Topological and functional studies on HlyB of Escherichia coli. Mol Gen Genet. 1992 Mar;232(1):40–48. [PubMed]
  • Gunderson KL, Kopito RR. Effects of pyrophosphate and nucleotide analogs suggest a role for ATP hydrolysis in cystic fibrosis transmembrane regulator channel gating. J Biol Chem. 1994 Jul 29;269(30):19349–19353. [PubMed]
  • Gunderson KL, Kopito RR. Conformational states of CFTR associated with channel gating: the role ATP binding and hydrolysis. Cell. 1995 Jul 28;82(2):231–239. [PubMed]
  • Guzman LM, Belin D, Carson MJ, Beckwith J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol. 1995 Jul;177(14):4121–4130. [PMC free article] [PubMed]
  • Higgins CF, Hiles ID, Salmond GP, Gill DR, Downie JA, Evans IJ, Holland IB, Gray L, Buckel SD, Bell AW, et al. A family of related ATP-binding subunits coupled to many distinct biological processes in bacteria. Nature. 1986 Oct 2;323(6087):448–450. [PubMed]
  • Hiraga S, Niki H, Ogura T, Ichinose C, Mori H, Ezaki B, Jaffé A. Chromosome partitioning in Escherichia coli: novel mutants producing anucleate cells. J Bacteriol. 1989 Mar;171(3):1496–1505. [PMC free article] [PubMed]
  • Horst M, Hilfiker-Rothenfluh S, Oppliger W, Schatz G. Dynamic interaction of the protein translocation systems in the inner and outer membranes of yeast mitochondria. EMBO J. 1995 May 15;14(10):2293–2297. [PMC free article] [PubMed]
  • Hwang S, Jascur T, Vestweber D, Pon L, Schatz G. Disrupted yeast mitochondria can import precursor proteins directly through their inner membrane. J Cell Biol. 1989 Aug;109(2):487–493. [PMC free article] [PubMed]
  • Koronakis E, Hughes C, Milisav I, Koronakis V. Protein exporter function and in vitro ATPase activity are correlated in ABC-domain mutants of HlyB. Mol Microbiol. 1995 Apr;16(1):87–96. [PubMed]
  • Koronakis V, Hughes C. Bacterial signal peptide-independent protein export: HlyB-directed secretion of hemolysin. Semin Cell Biol. 1993 Feb;4(1):7–15. [PubMed]
  • Koronakis V, Koronakis E, Hughes C. Isolation and analysis of the C-terminal signal directing export of Escherichia coli hemolysin protein across both bacterial membranes. EMBO J. 1989 Feb;8(2):595–605. [PMC free article] [PubMed]
  • Koronakis V, Hughes C, Koronakis E. Energetically distinct early and late stages of HlyB/HlyD-dependent secretion across both Escherichia coli membranes. EMBO J. 1991 Nov;10(11):3263–3272. [PMC free article] [PubMed]
  • Koronakis V, Hughes C, Koronakis E. ATPase activity and ATP/ADP-induced conformational change in the soluble domain of the bacterial protein translocator HlyB. Mol Microbiol. 1993 Jun;8(6):1163–1175. [PubMed]
  • Koronakis V, Li J, Koronakis E, Stauffer K. Structure of TolC, the outer membrane component of the bacterial type I efflux system, derived from two-dimensional crystals. Mol Microbiol. 1997 Feb;23(3):617–626. [PubMed]
  • Kübrich M, Dietmeier K, Pfanner N. Genetic and biochemical dissection of the mitochondrial protein-import machinery. Curr Genet. 1995 Apr;27(5):393–403. [PubMed]
  • Létoffé S, Delepelaire P, Wandersman C. Protein secretion in gram-negative bacteria: assembly of the three components of ABC protein-mediated exporters is ordered and promoted by substrate binding. EMBO J. 1996 Nov 1;15(21):5804–5811. [PMC free article] [PubMed]
  • Pugsley AP. The complete general secretory pathway in gram-negative bacteria. Microbiol Rev. 1993 Mar;57(1):50–108. [PMC free article] [PubMed]
  • Schatz G, Dobberstein B. Common principles of protein translocation across membranes. Science. 1996 Mar 15;271(5255):1519–1526. [PubMed]
  • Schülein R, Gentschev I, Mollenkopf HJ, Goebel W. A topological model for the haemolysin translocator protein HlyD. Mol Gen Genet. 1992 Jul;234(1):155–163. [PubMed]
  • Schülein R, Gentschev I, Schlör S, Gross R, Goebel W. Identification and characterization of two functional domains of the hemolysin translocator protein HlyD. Mol Gen Genet. 1994 Oct 28;245(2):203–211. [PubMed]
  • Stanley P, Koronakis V, Hughes C. Mutational analysis supports a role for multiple structural features in the C-terminal secretion signal of Escherichia coli haemolysin. Mol Microbiol. 1991 Oct;5(10):2391–2403. [PubMed]
  • Studier FW, Moffatt BA. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. [PubMed]
  • Voos W, von Ahsen O, Müller H, Guiard B, Rassow J, Pfanner N. Differential requirement for the mitochondrial Hsp70-Tim44 complex in unfolding and translocation of preproteins. EMBO J. 1996 Jun 3;15(11):2668–2677. [PMC free article] [PubMed]
  • Wagner W, Vogel M, Goebel W. Transport of hemolysin across the outer membrane of Escherichia coli requires two functions. J Bacteriol. 1983 Apr;154(1):200–210. [PMC free article] [PubMed]
  • Wandersman C, Delepelaire P. TolC, an Escherichia coli outer membrane protein required for hemolysin secretion. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4776–4780. [PMC free article] [PubMed]
  • Wickner WT, Lodish HF. Multiple mechanisms of protein insertion into and across membranes. Science. 1985 Oct 25;230(4724):400–407. [PubMed]
  • Zhang F, Sheps JA, Ling V. Complementation of transport-deficient mutants of Escherichia coli alpha-hemolysin by second-site mutations in the transporter hemolysin B. J Biol Chem. 1993 Sep 15;268(26):19889–19895. [PubMed]

Articles from The EMBO Journal are provided here courtesy of The European Molecular Biology Organization


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...