Figure 6.

In vitro binding between the SNARE complex and mutant Sec1p. Sec1p-V5-His₆ protein from wild-type (SEC1), class A mutant sec1-34, and class B mutant sec1-58 was affinity purified from yeast lysates by using an α-V5 antibody and immobilized on protein G-Sepharose resin for binding studies. The cytoplasmic domains of binary (Sso1p[1-265] and Sec9[416-651]) or ternary (Sso1p[1-265], Sec9p[416-651], and Snc2p[1-93]) SNARE complexes were incubated at 300 nM with immobilized mutant and wild-type Sec1p, as described previously (Togneri et al., 2006). Bound Sec1p-V5-His₆ and SNAREs were resolved by SDS-PAGE and stained using Coomassie Blue R250. Sec1-58p-V5-His₆ migrates slightly slower than Sec1p-V5-His₆ from the other strains. An asterisk marks a band likely to be a degradation product of Sec1p-V5-His₆ that copurify with the protein from the mutant strains. At higher SNARE complex concentrations, but not observed in this gel, the Snc2p band is detected at the position marked (Snc2p[1-93]; see Supplemental Figure S5). A diffuse band detected slightly above the Snc2p[1-93] position is a contaminant. V5 antibody heavy chain (Hc) and light (Lc) are also indicated. The positions of molecular weight standards are indicated in kilodaltons.