Extended Data Fig. 5: Cdt1’s calcium pump-independent function in fluconazole tolerance depends on its localization to the plasma membrane.

a Spotting assays for the indicated strains were performed on YPD plates supplemented with 0 or 0.4 M CaCl₂ as described in Extended Data Fig. 3a. Images were taken after 48 h of growth at 30 °C. The experiment was repeated twice with similar results. b Effect of the K603E substitution in Cdt1 on its plasma membrane localization. The indicated strains expressing GFP-Cdt1 or GFP-Cdt1^K603E were grown overnight in YPD and then subcultured at a 1:100 dilution into YPD with 0 or 30 µg/mL fluconazole. Images were taken after 4 h of growth at 30 °C. Scale bar, 5 μm. The experiment was repeated twice with similar results. c Quantification of the fluorescence intensity of GFP-Cdt1 at the plasma membrane observed in panel (b). Box plot format is the same as described in Fig. 3g. n = 50 for both WT and cdt1Δ:P_CDT1-CDT1^K603E cells. Statistical differences were determined by two-tailed, unpaired Student’s t-test. d Dose-response assays for the indicated strains were performed as in Fig. 3a. Fluconazole was applied as 2-fold serial dilutions ranging from 0.25 to 256 µg/mL. After incubation at 30 °C for 48 h, growth was measured and normalized to the no-drug control. Data are presented as mean ± SD of technical triplicates. Statistical differences were determined by two-way ANOVA multiple comparison test with the Geisser-Greenhouse correction. The experiment was repeated twice with similar results. e Corresponding MIC₅₀ and SMG levels calculated from data in panel (d). MIC₅₀ and SMG were calculated as described in Fig. 3b.

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