title: 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.
abstract: 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.
authors.affiliation: Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK.
authors.forename: T
authors.initials: T
authors.lastname: Thanabalu
authors.forename: E
authors.initials: E
authors.lastname: Koronakis
authors.forename: C
authors.initials: C
authors.lastname: Hughes
authors.forename: V
authors.initials: V
authors.lastname: Koronakis
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D001692:Biological Transport; D001704:Biopolymers; D002352:Carrier Proteins; D002462:Cell Membrane / Q000201:enzymology; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins / Q000378:metabolism*; D006868:Hydrolysis; D007425:Intracellular Membranes / Q000201:enzymology; D008565:Membrane Proteins; D026901:Membrane Transport Proteins
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D001704:Biopolymers; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C065231:HlyD protein, E coli; C036507:Hlya protein, E coli; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C073126:tolC protein, E coli; D000251:Adenosine Triphosphatases