Figure 5.

Mutations in a conserved groove disrupt Sec1p binding to the SNARE complex. (A) The positions of surface-directed mutations are mapped on nSec1. Mutations were introduced at three surfaces, indicated with dashed lines: the cleft, sec1-79 [E291K, E297R, D360R, D362K] (green spheres) and pCC72 [R63D, K64E] (black spheres); the furrow, sec1-59[G380R, E381K, D429K] (yellow spheres); and the groove sec1-58[K79E, Y80E, R169E, K170E] (blue spheres). The box (left; ribbon representation) highlights the groove region, where amino acids (blue spheres) are altered in class B mutants sec1-35 [N167I, F221Y, F235L], sec1-36 [I249N], sec1-46 [F156S, I187N], and sec1-48 [N81S, R638P] (see Table 2) and the site-directed groove surface mutant sec1-58[K79E, Y80E, R169E, K170E] (bold type). For labeling, amino acid numbers are based on the Munc18-1/nSec1 sequence, with the corresponding yeast numbers and mutations in parentheses. Images were created as described in Figure 1. (B) Groove mutants have a ts growth defect. Wild-type (SEC1) and sec1 mutants of the cleft (sec1-79), furrow (sec1-59), and groove (sec1-35, sec1-36, sec1-46, sec1-48, and sec1-58) were tested for growth at 25 and 38°C. A yeast strain bearing the plasmid pCC72 [R63D, K64E] in place of SEC1 was inviable. (C) Sec1p from the groove mutants binds weakly to assembled SNARE complexes. Wild-type (SEC1) and ts sec1 mutants (sec1-35, sec1-36, sec1-46, sec1-48, and sec1-58) were assayed for assembled SNARE complexes (top) and SNARE complex binding (middle), as described in Figure 2A. Input (bottom) represents 0.75% of the lysate used for the IPs.