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

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

^*Unité de Génétique Moléculaire, Centre National de la Recherche Scientifique URA 1773, 25, rue du Dr. Roux, Institut Pasteur, 75724 Paris Cedex 15, France; ^†Department of Crystallography, Birkbeck College, University of London, Malet Street, London WC1E 7HX, United Kingdom; ^‡Maurice E Müller-Institut, Biozentrum, University of Basel, Klingelbergstrasse 70, CH4056 Basel, Switzerland; and ^§Laboratoire des Biomembranes, Centre National de la Recherche Scientifique UMR 5619, Bat. 430, Université Paris Sud, 91405 Orsay Cedex, France

Communicated by William T. Wickner, Dartmouth Medical School, Hanover, NH (received for review February 5, 1999)

Abstract

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.

Footnotes

↵ ¶ To whom reprint requests should be addressed. e-mail: max{at}pasteur.fr.
ABBREVIATIONS:

EM,

electron microscopy;

STEM,

scanning transmission EM;

3D,

three-dimensional;

SB3–14,

3-(N,N-dimethylmyristylammonio)propanesulfonate