FIG. 5.

Yif1 is mislocalized to the vacuole in btn2Δ, rhb1Δ, vps26Δ, snx4Δ, chs4Δ, tlg2Δ, and ypt6Δ cells. (A) GFP-Yif1 is mislocalized to the vacuole in btn2Δ, chs4Δ, rhb1Δ, snx4Δ, tlg2Δ, vps26Δ, and ypt6Δ cells. A single-copy plasmid expressing GFP-Yif1 (pRS316-GFP-YIF1) was transformed into wild-type (WT) cells (both Euroscarf and W303-1b strains tested; Euroscarf cells shown) or cells bearing mutations of BTN2 (both Euroscarf and RKY4 btn2Δ strains tested; Euroscarf cells shown), RHB1 (both Euroscarf and JU28-1 rhb1Δ strains examined; Euroscarf strain shown), CHS4, VPS26, YPT6, SNX4, TLG2 VAM6, END4 (RH268-1c), RIC1, VPS51, and SEC7 (RSY979). Cells were grown to mid-log phase at 26°C on selective synthetic medium prior to pulse-labeling with FM4-64 (5.4 μM final concentration; 30 min at 26°C) and chase (30 min at 26°C) before visualization using confocal microscopy. sec7-5 (RSY979) cells were grown to mid-log phase at 26°C (permissive temperature) and either maintained and labeled with FM4-64 at 26°C or shifted to 37°C for 30 min prior to pulse-labeling with FM4-64 (30 min; 37°C) and chase (30 min; 37°C) before visualization. Note labeling of the vacuole by GFP in btn2Δ, chs4Δ, rhb1Δ, snx4Δ, tlg2Δ, vps26Δ, and ypt6Δ cells. See Table 3 for statistics regarding either Golgi or vacuolar GFP-Yif1 localization in the various strains examined. Merge indicates the merger of the GFP and FM4-64 fluorescence windows. (B) Btn2 and Btn2-GFP restore RFP-Yif1 localization to btn2Δ cells. btn2Δ cells expressing RFP-Yif1 from a multicopy plasmid (pAD54-RFP-YIF1) were transformed with either a control plasmid (pRS313) or single-copy plasmids expressing Btn2 or Btn2-GFP (pRS313-BTN2 or pRS313-BTN2-GFP). Cells were grown to mid-log phase at 26°C prior to visualization. Merge indicates the merger of light and fluorescence microscopy images.