Secretin PulD: Association with pilot PulS, structure, and ion-conducting channel formation

The outer membrane protein PulD (secretin) of Klebsiella oxytoca is require d for transport of pullulanase across this membrane. We have purified a mul timeric PulD complex from an Escherichia coli strain expressing all the pro teins involved in pullulanase secretion. The outer membrane-anchored lipopr otein PulS was found to copurify with PulD. The molar ratio of the two prot eins is close to 1:1, and the size of the complex is approximate to 1 MDa. Scanning transmission electron and cryo-electron microscopy analyses showed that the purified complex is a cylindrical structure having a central cavi ty of approximate to 7.6 nm and peripheral radial spokes. Fusion of proteol iposomes containing the purified complex with a planar lipid bilayer result ed 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 fluctuation s correspond to voltage-induced, relatively minor displacements of domains in the purified complex rather than to a complete opening of the secretin c hannel.