Extended Data Fig. 4: Calcineurin promotes Cdt1’s calcium pump-independent function through a post-transcriptional mechanism.

a Calcium-binding loops in C. auris calmodulin, CMD1. Four potential calcium-binding loops were predicted based on the structure of S. cerevisiae calmodulin⁸⁵. The residues highlighted in red were mutated (D to A and E to V) to abolish its calcium-binding activity, yielding CMD1-4. b Spotting assays for the indicated strains were performed on YPD plates supplemented with 0, 0.4, 0.6, or 0.8 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. c Domain organization of the catalytic subunit of C. auris calcineurin, calcineurin A (CNA1). Catalytic, the phosphatase domain; CNB-binding, calcineurin B subunit domain; CAM-binding, calmodulin-binding domain; AI, the auto-inhibitory domain. The boundaries of these domains are defined according to the structure of the rat calcineurin A α-isoform⁷⁰. Two stop codons were introduced at L453 to generate a constitutively activated calcineurin A, CNA1tr. d Dose-response assays for the indicated strains were performed as described in Fig. 3a. Fluconazole was applied as 2-fold serial dilution 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. f qPCR analysis of CDT1 transcript levels. The indicated strains were grown overnight in YPD and then subcultured at a 1:100 dilution into YPD containing 0 or 30 µg/mL fluconazole. Cells were collected for RNA extraction after 4 h of growth at 30 °C. CDT1 expression levels were normalized to ACT1, with the level of the untreated WT strain set to 1. Data are presented as mean ± SD of technical triplicates. Statistical differences were determined by two-tailed, unpaired Student’s t-test. The experiment was repeated twice with similar results. g Dose-response assays for the indicated strains were performed as described in Fig. 3a. Fluconazole was applied as 2-fold serial dilution 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. h Corresponding MIC₅₀ and SMG levels calculated from data in panel (g). MIC₅₀ and SMG were calculated as described in Fig. 3b. i The calcium-calcineurin signaling pathway drives Cdt1’s plasma membrane localization post-transcriptionally. The indicated strains expressing GFP-Cdt1 under the ADH1 promoter 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.

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