The seven functionally distinct serotypes (A-G) of botulinum neurotoxin (BoNT) are dichains consisting of light chain (LC) with zinc-dependent endoprotease activity connected by one disulfide bond to heavy chain with neuronal-cell translocation and receptorbinding domains. LC-mediated proteolysis of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and consequent inhibition of synaptic vesicle fusion to the presynaptic membrane of human motor neurons are responsible for flaccid paralysis associated with botulism. LC endoproteolysis is complex, requiring highly extended SNARE sequences at the surface of intracellular membranes and prompting our development of a genetically amenable assay to monitor the interaction between BoNT͞LC and its SNARE substrate. Using BoNT serotype B as a model, the assay employs a chimeric SNARE protein where a portion of neuronal synaptobrevin (Sb) is fused to Snc2p, a Sb ortholog required for protein secretion from yeast cells. Regulated expression of serotype B-LC in yeast leads to cleavage of the chimera and a conditional growth defect. To assess utility of this assay for monitoring SNARE protein cleavage, we growth-selected chimeric SNARE mutations that inhibited proteolysis. When these mutations were introduced into Sb and examined for cleavage, substrate residues located near and distal to the cleavage site were important, including residues positioned near the Sb transmembrane domain, an unexplored aspect of BoNT cell intoxication. Additional mutations were positioned in a nine-residue SNARE motif, supporting a previously assigned role for this motif in LC recognition and providing proof of principle for the application of yeast-based technology to study intracellular BoNT͞LC endoproteases.substrate specificity ͉ endoprotease ͉ endopeptidase ͉ synaptic vesicle B otulinum neurotoxin (BoNT) intoxicates motor neurons at neuromuscular junctions to produce a generalized flaccid paralysis that is often fatal at extremely low doses of the toxin: LD 50 values of 1-5 ng͞kg in mice (1). The potency of BoNT has caused its listing as a class A select toxin by the Centers for Disease Control and Prevention, the National Institute of Allergy and Infectious Disease, and the U.S. Department of Agriculture. There are seven structurally related BoNT serotypes (A-G), each possessing a heavy chain capable of transporting its light chain (LC) partner into human neuronal cells. Upon entering the cytoplasmic compartment, LC endoprotease cleaves and inactivates one of three membrane-bound soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, including one vesicle SNARE, synaptobrevin (Sb), and two target membrane SNAREs, SNAP-25 and syntaxin. These SNARE proteins physically interact in a calcium-dependent manner, leading to fusion of synaptic vesicles to the presynaptic membrane of motor neurons (Fig. 1a). BoNT͞LC disables the physical interaction between SNARE proteins, inhibiting vesicle fusion and acetylcholine release into the n...