Fig 4. zrt1Δ cells accumulate lower levels of zinc in the cytosol.

(A) zrt1Δ cells expressing ZapCY1 were grown overnight in ZL-EMM. At t = 0 cells were exposed to 0–1000 μM. Total cellular zinc was then determined by AAS as described for Fig 3A. (B-F) zrt1Δ cells expressing ZapCY1 or ZapCY2 were grown overnight in ZL-EMM. Cells were transferred to temperature-controlled cuvettes and at t = 0 shocked with 0–1000 μM Zn²⁺. Changes in the FRET ratio were measured as described in Fig 3B. Panels B and D show representative experiments and panels C and E show the average values from 3 independent experiments with error bars representing standard deviations. Panel F shows a comparison of a 1 μM zinc shock in wild-type and zrt1Δ cells expressing ZapCY1 (G-I) Wild-type and zrt1Δ cells expressing ZapCY1 or ZapCY2 were grown overnight in ZL-EMM. At t = 0 cells were shocked with 100 μM Zn²⁺. Cells were harvested at the indicated time points and crude protein extracts prepped for immunoblot analysis. Immunoblots were incubated with antibodies to GFP and loading control Act1.