Single molecule detection of intermediates during botulinum neurotoxin translocation across membranes.
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.
Fischer
Audrey
A
Section of Neurobiology, Division of Biological Sciences, University of California at San Diego, La Jolla, CA 92093-0366, USA.
Montal
Mauricio
M
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D001905:Botulinum Toxins / Q000737:chemistry / Q000378:metabolism*; D019274:Botulinum Toxins, Type A / Q000737:chemistry / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D003014:Clostridium botulinum / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D018832:Molecular Chaperones / Q000378:metabolism; D009447:Neuroblastoma / Q000473:pathology; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D009498:Neurotoxins / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D018408:Patch-Clamp Techniques; D011489:Protein Denaturation; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D020673:Protein Renaturation; D021381:Protein Transport; D012680:Sensitivity and Specificity; D013997:Time Factors
D004220:Disulfides; D007140:Immunoglobulin Fab Fragments; D018832:Molecular Chaperones; D009498:Neurotoxins; D020033:Protein Isoforms; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; C096326:botulinum toxin type E