Figure 2. LegC7 induces endosome:vacuole trafficking defects.

(A) BY4742 yeast strains harboring GFP-CPS and either the vector control, LEGC7 ⁺, or LEGC7 ^N242I plasmids were grown in selective media supplemented with 2% glucose at 30°C, washed in ddH₂O, suspended in fresh CSM-uracil/2% galactose, incubated at 30°C for 16 h, then visualized. (B) Equal portions of total proteins were extracted from strains in (A), then immunoblotted for GFP and Sec17p (loading control). (C) BY4742 yeast strains containing GFP-Sna3 and either the vector control, LEGC7 ⁺, or LEGC7 ^N242I plasmids were grown as in (A), then visualized. (D) Cells containing the vector control, LEGC7 ⁺, or LEGC7 ^N242I plasmids were incubated with Lucifer Yellow (Materials and Methods), and then visualized. (E) Strains from (D) were grown in selective media supplemented with 2% glucose at 30°C, washed in ddH₂O, suspended in fresh CSM-uracil/2% galactose, incubated at 30°C for 16 h, then stained with the yeast vacuolar marker FM4–64 [57] and visualized. (F) Wild type SEY6210 or ∆4+ENTH (Table 1) strains harboring Ste3-GFP and either the vector control or LEGC7 ⁺ plasmids were grown as in (A) and then visualized.