Figure 7: Disruption of the Sec3–Sso interaction in yeast leads to exocytosis defects.
From: Sec3 promotes the initial binary t-SNARE complex assembly and membrane fusion
(a) His6-Sso2 was incubated with GST-tagged wild-type or mutant Sec3N for binding assay. The sec3N-mutS1 and sec3N-mutS2 mutants showed much reduced binding to Sso2. (b) SEC3, sec3ΔN, sec3-mutS1 and sec3-mutS2 were expressed under the endogenous SEC3 promoter in the sec3Δ background. The yeast cells were grown on synthetic complete plates at 25 and 37 °C. The sec3 mutants show growth defects at 37 °C. (c) Analysis of Bgl2 secretion for the above yeast strains. Yeast cells were grown at 25 °C before being transferred to 37 °C for 2 h. Internal and external Bgl2 was detected by western blotting. Alcohol dehydrogenase-1 (Adh1) was used as a loading control. (d) Invertase secretion from yeast cells of the above strains at 25 and 37 °C. The percentage of invertase secretion was plotted. The values in the graph represent the average of three experiments. Student’s t-test was used for statistical analyses. Error bars: s.e. ‘*’P<0.05. (e) Thin-section electron microscopy analysis of cells expressing SEC3, sec3ΔN, sec3-mutS1 and sec3-mutS2. Cells were fixed with permanganate, and the vesicles were shown in dark staining. An accumulation of secretory vesicles was detected in cells expressing Sec3 mutants. Scale bar, 0.5 μm.
