FIGURE 4: Yft2-GFP mainly escapes the ER to reach the vacuole. (A) Scs3-GFP expressed from the PHO5 promoter. (B) The same construct expressed from the SCS3 pro-moter. Scs3-GFP localises to the ER, with diagnostic nuclear profiles (arrowheads), and only a faint signal with the endogenous promoter. (C) Yft2-GFP expressed from the PHO5 promoter. In addition to some nuclear profiles, there are large spherical regions of diffuse staining (intravacuole – asterisks) and additional puncta. (D) Yft2-GFP expressing cells were incubated with FM4-64 on ice to label the plasma membrane, then warmed to 30 °C for 10 minutes to allow dye to enter the endocytic pathway, but not to reach vacuoles. Puncta positive for Yft2-GFP (left hand panel) that were also positive for FM4-64 (middle panel) are indicated by arrowheads, and the degree of overlap is also shown in a merged coloured image (right hand panel, GFP = green, FM4-64 = magenta, overlap = white).