Figure 3. Vps41 and Vps39 differ in vacuole affinity and co-localization with Ypt7. (A, B) Vps41 and Vps39 relocalize more efficiently in the absence of Ypt7. FRB-GFP tagged Vps41 (A) and Vps39 (B) were observed as in Figure 2 by fluorescence microscopy in the indicated strains. To resolve their dynamic relocalization over time, 100 nM rapamycin was added to the cells. (C-H) Vps41 and Vps39 differ in their Ypt7-interaction on vacuoles as revealed by the Split-YFP approach. Ypt7 was tagged with the C-terminal half of Venus (VC) in all strains, whereas Vps39 and Vps41 were tagged N-terminally (C, E) or C-terminally (D, F) with the VN part.³⁴ For colocalization analysis, the cells expressing VC-Ypt7 and VN-Vps39 were additionally modified by tagging Vps41 with mCherry (G) or transformed with a CEN plasmid expressing dsRED-Ypt7 under the control of a PHO5 promoter (H). All cells were observed by fluorescence microscopy. Scale bar, 5 µM.