Vacuolar Zn²⁺ transporters can be downregulated in response to changes in Zn²⁺ concentration. (A) A schematic diagram illustrating the topology of vacuolar Zn²⁺ transporters, including Zrc1, Cot1, and Zrt3. (B) Localization of chromosomally tagged Zrc1-GFP, Cot1-GFP, Zrt3*-GFP and Vph1-mCherry, before and 6 h after Zn²⁺ withdrawal in YNB media. Dashed lines highlight the cell surface. (C) Western blot analysis of chromosomally tagged Zrc1-GFP, Cot1-GFP, and Zrt3*-GFP over the course of 6 h of Zn²⁺ withdrawal in YNB media. The samples were blotted with an anti-GFP antibody. G6PDH was used as a loading control. Same volume of cells was loaded in each lane, with 1 OD₆₀₀ cells loaded at 0 h. (D) Quantification of 1C. All protein levels were normalized using G6PDH level. (E) Localization of Zrt3*-GFP under different Zn²⁺ treatments. Mid-log cells grown in YPD (0 h) were transferred into YNB without Zn²⁺ and incubated for 3 h (−Zn²⁺ 3 h). Then, 2 mM ZnCl₂ was added and cells were incubated for another 3 h (+Zn²⁺ 3 h). (F) Western blot analysis of Zrt3*-GFP and Vph1 levels when yeast cells were shifted from YNB without Zn²⁺ to YNB with excessive Zn²⁺ (2 mM ZnCl₂). G6PDH was used as a loading control. 1 OD₆₀₀ cells were loaded in each lane. (G) Quantification of F. All protein levels were normalized using G6PDH level. (H) Localization of tet-O7 Cot1-GFP and Vph1-mCherry before and after Zn²⁺ withdrawal in YNB media. The incubation was performed in the presence of 2 µg/ml doxycycline (Dox). Right: Line-scan analysis of the image. (I) Degradation kinetics for tet-O7 Cot1-GFP after addition of 2 µg/ml Dox and Zn²⁺ withdrawal in YNB media. G6PDH was used as a loading control. Same volume of cells were loaded, with 1 OD₆₀₀ cells loaded at 0 h. (J) Quantification of I. All protein levels were normalized using G6PDH level. Bar, 1 µm.