Figure 4. C. neoformans MTs are Cu responsive Cu detoxification proteins (See also Table S1).
(A). C. neoformans cell growth assays were performed in SC medium in 96 well plates. Cell cultures of wildtype and cmt1Δ cmt2Δ were diluted to an A600 of 0.002, supplemented with metals. A600 was measured after overnight growth. ΔA600 was calculated by subtracting from blank (medium without cells). Graphs show average of three biological replicates. Error bars indicate SD.
(B). C. neoformans metal sensitivity assays on agar medium. Cell cultures of wild type and cmt1Δ cmt2Δ cells were diluted in water to an A600 of 1.0. 10-fold serial dilutions cells were spotted onto SC agar or agar supplemented with 400 µM Cu, Zn, Fe, 100 µM Cd, 40 µM BPS, or 10 µg/ml menadione. Plates were incubated for 2 days (6 days for menadione spotting assay) and photographed.
(C). Expression of CMT1 and CMT2 was quantitated by qRT-PCR. Cell cultures were diluted to an A600 of 0.2 in SC medium at 37°C for 1hr, supplemented with the indicated concentrations (selected according to the results from Figure 4A. 10, 100, 1000 µM For Cu, Zn, Fe; 5, 10, 20 µM for Cd; 10, 20, 40 µM for BPS; 0.2, 0.4, 0.8 µg/ml for menadione). Error bars indicate SD.
(D). Protein expression of Cmt1-FLAG was confirmed by immunoblotting. Cells were grown as described as Figure 4C. Protein extracts were treated with TCEP, resolved by SDS-PAGE and anti-FLAG mouse antibody was used for immunoblotting. Coomassie staining was used as a loading control.