Figure 4.

Both Tul1 and Rsp5 contribute to the degradation of Zrt3*-GFP. (A) The internalization of Zrt3*-GFP into the vacuole lumen was not blocked in ssh4Δ strain after the addition of 2mM ZnCl₂ to the YNB media. Cells were pretreated with YNB without Zn²⁺ for 3 h. (B) Degradation kinetics for Zrt3*-GFP in WT and ssh4Δ strains. G6PDH was used as a loading control. Same volume of cells was loaded, with 1 OD₆₀₀ cells loaded at 0 h. (C) Localization of Zrt3*-GFP in WT, tul1Δ, rsp5-1, and tul1Δ rsp5-1 mutant strains before and after addition of 2mM ZnCl₂ to YNB media. Cells were pretreated with YNB without Zn²⁺ for 3 h at 26°C. Then, the culture was shifted to 37°C for 30 min before the addition of ZnCl₂ and the incubation was continued for another 3 h at 37°C. (D) Degradation kinetics for Zrt3*-GFP in WT, tul1Δ, rsp5-1, and tul1Δ rsp5-1 mutant strains. The cells were pretreated with YNB media without Zn²⁺ for 3 h at 26°C. Then, the culture was shifted to 37°C for 30 min before the addition of ZnCl₂ to 2 mM and the incubation was continued for another 3 h at 37°C. G6PDH was used as a loading control. Same volume of cells was loaded, with 1 OD₆₀₀ cells loaded at 0 h. Bar, 1 µm.