Secretin PulD: association with pilot PulS, structure, and ion-conducting channel formation.
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 approximately 1 MDa. Scanning transmission electron and cryo-electron microscopy analyses showed that the purified complex is a cylindrical structure having a central cavity of approximately 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.
Unité de Génétique Moléculaire, Centre National de la Recherche Scientifique URA 1773, 25, rue du Dr. Roux, Institut Pasteur, 75724 Paris Cedex 15, France.
N
N
Nouwen
N
N
Ranson
H
H
Saibil
B
B
Wolpensinger
A
A
Engel
A
A
Ghazi
A P
AP
Pugsley
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D003001:Cloning, Molecular; D020285:Cryoelectron Microscopy; D004926:Escherichia coli; D006026:Glycoside Hydrolases / Q000096:biosynthesis; D007091:Image Processing, Computer-Assisted; D007473:Ion Channels / Q000502:physiology*; D007709:Klebsiella / Q000502:physiology*; D008051:Lipid Bilayers; D008564:Membrane Potentials; D017348:Microscopy, Electron, Scanning Transmission; D008958:Models, Molecular; D011510:Proteolipids
D001425:Bacterial Outer Membrane Proteins; D007473:Ion Channels; D008051:Lipid Bilayers; D011510:Proteolipids; C020485:proteoliposomes; C061377:PulD protein, Klebsiella pneumoniae; D006026:Glycoside Hydrolases; C100181:pullulanase