Figure 2.

Phenotypes of class A and class B sec1 mutants. (A) SNARE complex abundance is diminished in class A mutants, whereas SNARE complex binding to Sec1p is disrupted in class B mutants. An α-Sec9p antibody (Sec9-288) was used to IP Sec9p in an assay for assembled SNARE complexes (top). An α-Sec1p antibody (Sec1-229) was used to IP Sec1p in an assay for Sec1p-binding to SNARE complexes (middle). Coprecipitation of Sso1p with either Sec9p or Sec1p was detected by Western blot analysis by using an α-Sso1p antibody (Sso1-16,371). Wild-type (SEC1) and the indicated sec1, sec6, and exo70 mutant yeast strains were lysed after a 20-min shift from permissive (25°C) to restrictive (38°C) temperature. Protein concentration in the lysates was determined using the Bradford assay (Bio-Rad Laboratories). Bound proteins were separated by SDS PAGE, and 0.75% of the lysates used for the IPs is shown (Input, bottom). (B) Both invertase and Bgl2p secretion are tightly blocked in class A mutants and blocked with a range of severity in class B mutants. Accumulation of invertase and Bgl2p was measured in wild-type (SEC1) and the indicated mutant yeast strains after a 60-min shift to the restrictive temperature (38°C). Invertase activity was measured using an enzyme assay, and the average percentage of internal invertase (±SEM; n = 4) was plotted on a bar graph (black; see also Supplemental Figure S2). The average internal Bgl2p was measured by Western blot analysis by using an α-Bgl2p antibody (gray; for Westerns, see Supplemental Figure S2). The averaged units of intensity plotted (±SEM; n = 3) represent the optical density per square millimeter of each Bgl2p band, normalized to the intensity of internal loading control (ADH).