A yeast assay probes the interaction between botulinum neurotoxin serotype B and its SNARE substrate

Abstract

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 receptor-binding 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 BoNT/LC 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 BoNT/LC endoproteases.

Fig. 1.

Neuronal and yeast SNAREs. (a) Vesicle-associated SNAREs Sb (human neuronal cells) and Snc (yeast cells) physically interact with target membrane SNAREs SNAP25 and syntaxin (neurons) and Sec9 and Sso (yeast), leading to secretion of neurotransmitters across the presynaptic membrane (neurons) and proteins across the plasma membrane (yeast). (b) Relevant protein sequences of SNARE proteins Snc2, Sb, and Snc2/Sb/Snc2 are depicted. Sb2 sequences are in boldface. The minimal Sb sequence required for efficient B-LC cleavage (3) is enclosed by brackets. The B-LC cleavage site is indicated by the slashes. The two SNARE motifs in Sb2 (8) are underlined. Sequences of probable transmembrane segments are indicated by italics. Positions and identities of amino acid substitutions are depicted.

Fig. 2.

Development of yeast assays for B-LC. (a) Yeast strains BY4741 (SNC2 Δsnc1)/pHF456 (URA3)/pBLC1 (GAL1-B-LC HIS3) and HFY801 (Δsnc1 Δsnc2)/pHF550 (ADH1-Snc2/Sb/Snc2 URA3)/pBLC1 (GAL1-B-LC HIS3) were placed on glucose agar plates (to repress expression of B-LC) and galactose agar plates (to derepress expression of B-LC). Plates were incubated at 30°C for 3 and 5 days, respectively. (b) WT yeast strains without B-LC, SEY6210.5/pRS314 (TRP1) and with B-LC, SEY6210.5/pBLCFLAG (TRP1) were grown to logarithmic phase on glucose and galactose, and cells were subjected to Western blotting as described in Methods. B-LC gene was expressed by the GALS promoter in galactose and not in glucose. (c) HA-Snc2/Sb/Snc2 cleavage was examined in cells of strain HFY801/pHF551 (ADH1-HA-Snc2/Sb/Snc2 HIS3) with and without pBLC2 (GAL1-B-LC URA3) by pulse labeling as described in Results. (d) Probable palmitoylation of HA-Snc2/Sb/Snc2 at residue C94 in strain HFY801 is indicated by the absence of a doublet in SDS/PAGE-displayed (C94S) mutant protein. (e) B-LC cleavage of HA-Sb in yeast strain SEY6210.5 (WT)/pWL21 (ADH1-HA-Sb TRP1) with and without pWL22 (HPT1-B-LC URA3) was examined by pulse labeling as described in Results.

Fig. 3.

Expression of chimeric SNARE mutations in yeast. (a) Yeast strain HFY801/pHF551 (HA-Snc2/Sb/Snc2) with (sectors 2–9) and without (sector 1) pBLC2 (B-LC) were placed on agar plates containing glucose (3 days) or galactose (5 days). Sectors 1 and 2 show nonmutated Snc2/Sb/Snc2, whereas sectors 3–9 show Snc2/Sb/Snc2 with yeast-selected mutations listed in b. (b) Yeast strains (from a) were examined by pulse labeling. Lane numbers correspond to sector numbers in a. Amount of cleavage product is indicated as a percentage of total HA-Snc2/Sb/Snc2 expressed during the pulse and represents an average from three independent trials. Background protein or degradation product is indicated by an asterisk.

Fig. 4.

Cleavage of Sb mutants in yeast. (a) Yeast strain SEY6210.5/pWL22 (B-LC) was transformed with a series of plasmids encoding nonmutated HA-Sb (WT) and the indicated HA-Sb mutants. Mutations in the gene encoding HA-Sb were constructed by site-directed mutagenesis using a two-step PCR mutagenesis approach (22). Sequences of oligonucleotide primers used to construct mutations can be found in Table 1, which is published as supporting information on the PNAS web site. Sequences of other primers used in this study are available on request. Transformed yeast cells were examined by pulse labeling as described in Results. Amount of cleavage product is indicated as a percentage of total HA-Sb expressed during the pulse and represents an average from two independent trials. (b) Translational stop codons were introduced into the cleavage sites (between Q76 and F77) of Sb mutants D64G, D65G, and Q71R. SEY6210.5 cells expressing these truncated products were subjected to 15-min pulse labeling, and proteins were analyzed by SDS/PAGE. Apparent size of the truncations was compared with apparent size of B-LC cleavage products from corresponding Sb mutants by using SDS/PAGE gels. Data reveal anomalous migration patterns of proteins with D64G, D65G, and Q71R substitutions.

Fig. 5.

Cell-free cleavage of Sb by B-LC. Individual mutations were introduced into Sb (1–96) by site-directed mutagenesis (see Methods), and purified proteins were subjected to cleavage with the indicated concentration of recombinant B-LC [LC/B]. The reaction was stopped and subjected to SDS/PAGE to detect Sb cleavage. The asterisk indicates the concentration of B-LC (nM) required to cleave 50% of Sb.