Figure 7
CT105 induces a vacuolar protein sorting defect in S. cerevisiae. S. cerevisiae reporter strain NSY01 producing CPY-invertase was transformed with plasmids encoding GFP (pKS84), VipA-GFP, GFP-CT105, or CT105-GFP, where their expression can be induced by galactose. A NSY01 derivative strain encoding a dominant-negative form of the yeast ATPase Vps4 (Vps4E233Q) was also used. Yeast strains are listed in Supplementary Table S2. (A) Whole cell extracts of S. cerevisiae NSY01 producing the indicated proteins were analyzed by immunoblotting with antibodies against GFP and PGK1 (yeast loading control), using SuperSignal West Pico detection kit (Thermo Fisher Scientific). (B) S. cerevisiae NSY01 strains encoding the indicated proteins were grown in solid medium in the presence of galactose (inducing conditions, +GAL) or in the presence of fructose (non-inducing conditions; +FRU). The vacuolar protein sorting (VPS) phenotype was analyzed using a sucrose overlay to assess activity of secreted invertase. A vacuolar protein sorting defect (VPS−) leads to the formation of a brown precipitate. (C) S. cerevisiae NSY01 strains encoding the indicated proteins were grown in liquid medium in the presence of galactose (inducing conditions) and the relative activity of secreted invertase was quantified (see Experimental Procedures). Data are mean ± standard error of the mean of five independent experiments. P-values were obtained by one-way ANOVA and Dunnett post-test analyses relative to GFP (0% of relative secreted invertase; not shown); *statistical significant (P < 0.05); ns, not significant.
