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J. Biol. Chem., Vol. 282, Issue 40, 29604-29611, October 5, 2007

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Crucial Role of the Disulfide Bridge between Botulinum Neurotoxin Light and Heavy Chains in Protease Translocation across Membranes^*

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

Clostridial botulinum neurotoxins (BoNTs) exert their neuroparalytic action by arresting synaptic exocytosis. Intoxication requires the disulfide-linked, di-chain protein to undergo conformational changes in response to pH and redox gradients across the endosomal membrane with consequent formation of a protein-conducting channel by the heavy chain (HC) that translocates the light chain (LC) protease into the cytosol. Here, we investigate the role of the disulfide bridge in the dynamics of protein translocation. We utilize a single channel/single molecule assay to characterize in real time the BoNT channel and chaperone activities in Neuro 2A cells under conditions that emulate those prevalent across endosomes. We show that the disulfide bridge must remain intact throughout LC translocation; premature reduction of the disulfide bridge after channel formation arrests translocation. The disulfide bridge must be on the trans compartment to achieve productive translocation of LC; disulfide disruption on the cis compartment or within the bilayer during translocation aborts it. We demonstrate that a peptide linkage between LC and HC in place of a disulfide bridge is insufficient for productive LC translocation. The disulfide linkage, therefore, dictates the outcome of translocation: productive passage of cargo or abortive channel occlusion by cargo. Based on these and previous findings we suggest a sequence of events for BoNT LC translocation to be HC insertion, coupled LC unfolding, and protein conduction through the HC channel in an N to C terminus orientation and ultimate release of the LC from the HC by reduction of the disulfide bridge concomitant with LC refolding in the cytosol.

Received for publication, May 1, 2007 , and in revised form, July 30, 2007.

^* This work was supported by the U.S. Army Medical Research and Materiel Command (DAMD17-02-C-0106), National Institutes of Health Training Grant T32 GM08326, and Pacific Southwest Regional Center of Excellence Grant AI-65359. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Section of Neurobiology, Division of Biological Sciences, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0366. Tel.: 858-534-0931; Fax: 858-822-3763; E-mail: mmontal{at}ucsd.edu.

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