Fig. 4: Random mutagenesis reveals a hotspot for residues important for Erg251 function under EHC.

a Strategy to identify erg251 BinGo mutants. Error-prone PCR pool of ERG251 fused with a NAT resistance marker was introduced into the “Parent” strain. Colonies that grew robustly on YPD + NAT + DOX under AC, but poorly under EHC were selected b Missense mutations on the prioritized BinGo alleles. Magenta, light green, or black, respectively, color-code major, minor, or insignificant improvement of EHC fitness when the indicated mutation was back-mutated. Grey: untested. c A predicted model of Erg251 suggests locational enrichment of mutations making major contributions to BinGo phenotypes. Sites highlighted magenta in (b) are marked. Yellow: transmembrane motifs predicted by TMAlphaFold (TM1 to TM5). Yellow dots count the number of times for major contributing mutations identified at a given residue. d Mutants sufficient to drive a notable BinGo phenotype and their predicted spatial position relative to Fe2. AC and EHC growth of WT and mutant strains were tested on YPD + 20 µg mL⁻¹ DOX. e Targeted mutagenesis identifies additional BinGo substitutions. Each mutant was expressed in the “Parent” background and growth on YPD + 20 µg mL⁻¹ DOX was assessed under both AC and EHC. f Spot dilution of erg25-deletion strains expressing mutant (homozygous substitution) or WT ERG251 on YPD under AC and EHC. AC growth was tested at both 37 °C and 30 °C to determine temperature-dependent defects. GC-MS analyses confirm comparable ergosterol content in WT and mutant strains. ns not significant (p-values:0.29; 0.11) g The native promoter-driven WT ERG251 or BinGo mutants was expressed in a background where one copy of ERG251 was deleted and the other controlled by a tetO promoter (tetO-ERG251/Δ). Strains were grown on SD + 20 µg mL⁻¹ DOX under EHC before collection for sterol profiling by GC-MS. Detailed information for (b, d, and e) are provided in Supplementary Figs. 3b and 5a, b. Sterol contents in (f and g) show mean ± SD of biological triplicates. Unpaired t-tests were performed and two-tailed p-values reported. Exact p-values in (g) were provided in Source Data.