Résumé
The outer membrane protein PulD (secretin) of Klebsiella oxytoca is required for transport of pullulanase across this membrane. We have purified a multimeric PulD complex from an Escherichia coli strain expressing all the proteins involved in pullulanase secretion. The outer membrane-anchored lipoprotein PulS was found to copurify with PulD. The molar ratio of the two proteins is close to 1:1, and the size of the complex is ≈1 MDa. Scanning transmission electron and cryo-electron microscopy analyses showed that the purified complex is a cylindrical structure having a central cavity of ≈7.6 nm and peripheral radial spokes. Fusion of proteoliposomes containing the purified complex with a planar lipid bilayer resulted in the appearance of small, voltage-activated, ion-conducting channels. We conclude that the central cavity seen in the electron microscope is part of a large gated channel and propose that the observed current fluctuations correspond to voltage-induced, relatively minor displacements of domains in the purified complex rather than to a complete opening of the secretin channel.