Fig. 3. Targeting of GFP by Vac8 wild-type and mutant SH4 domains in yeast. GFP-fusion proteins encoding the first 18 amino acids of Vac8 (A) or the indicated mutations (B) were expressed in BJ3505 vac8 cells and analyzed by fluorescence microscopy. Bars, 5 µm. (C) Subcellular localization of Vac8(1-18)-GFP. The indicated cells were analyzed as in Fig. 1C. Proteins were analyzed by SDS-PAGE and western blotting with anti-GFP antibodies. (D) Palmitoylation of the SH4 constructs. The biotin-switch method was used as described in Fig. 2D and Hou et al. (Hou et al., 2005). Western blots were decorated as in C.