Single molecule detection of intermediates during botulinum neurotoxin translocation across membranes

Audrey Fischer and
Mauricio Montal*

Section of Neurobiology, Division of Biological Sciences, University of California at San Diego, La Jolla, CA 92093-0366

Edited by Tom A. Rapoport, Harvard Medical School, Boston, MA, and approved March 30, 2007 (received for review January 3, 2007)

Abstract

The dynamics of Clostridium botulinum neurotoxins (BoNTs) protein-translocation across membranes was investigated by using a single molecule assay with millisecond resolution on excised patches of neuronal cells. Translocation of BoNT/A light chain (LC) by heavy chain (HC) was observed in real time as an increase of channel conductance: the HC channel is occluded by the LC during transit, then unoccluded after completion of translocation and release of LC-cargo. We identified an entirely unknown succession of intermediate conductance stages during LC translocation. For the single-chain BoNT/E, by contrast to the di-chain BoNT/A, we demonstrate that productive translocation requires proteolysis of the LC cargo from the HC chaperone. We propose a model for the set of protein–protein interactions between translocase and cargo at each step of translocation that supports the notion of an interdependent, tight interplay between the HC chaperone and the LC cargo preventing LC aggregation and dictating the outcome of translocation: productive passage of cargo or abortive channel occlusion by cargo.

Footnotes

*To whom correspondence should be addressed. E-mail: mmontal{at}ucsd.edu
Author contributions: A.F. and M.M. designed research; A.F. and M.M. performed research; M.M. contributed new reagents/analytic tools; A.F. and M.M. analyzed data; and A.F. and M.M. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0700046104/DC1.
Abbreviations:

γ,

single channel conductance;

BoNT,

botulinum neurotoxin;

LC,

light chain;

HC,

heavy chain;

Po,

channel open probability;

TCEP,

tris-(2-carboxyethyl) phosphine;

V½,

the voltage at which Po = 0.5;

SNARE,

soluble NSF attachment protein receptor.