Article Figures & Data
Figures
- FIG 1
Cap2 evolutionary conserved domains are essential for growth under iron-deprived conditions. (A) Schematic diagram of wild-type (WT) and mutant derivatives of Cap2. aa, amino acids; CTD, C-terminal domain. (B) Growth phenotype of the wild-type strain, and strains bearing the various N-terminal and C-terminal mutations in Cap2. The strains were precultured in SC-LIM liquid medium, serially diluted and spotted on SC-LIM alone or on SC-LIM plus BPS, SC-LIM plus BPS and FAS, or SC-LIM plus FAS.
- FIG 2
The C-terminal deletion mutant protein is expressed in iron-depleted medium. Whole-cell extracts from cap2Δ/Δ (lane 1), wild type (lanes 2 and 3), cap2-1 (lane 4), ΔC1 (lane 5), ΔC2 (lane 6), ΔC3 (lane 7), ΔC4 (lane 8), and cap2-2 (lane 9) strains were separated on 8 to 16% gradient SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and probed with affinity-purified anti-Cap2 antibody. A 72-kDa nonspecific band that consistently appeared in all lanes as reported previously (9) is indicated by an asterisk.
- FIG 3
Transcriptional regulation of iron homeostasis genes is impaired in the bZIP, HAP4L, and C-terminal domain mutants. (A to C) qRT-PCR analysis of the expression of Cap2-repressed ACO1 (A) and CYC1 (B) mRNA levels and Cap2-induced FRP1 mRNA levels (C) in wild-type and mutant strains. Fold change was calculated from three independent experiments, and Student’s t test was conducted to determine statistical significance. The values for mutants that are significantly different from the value for the wild type are indicated by asterisks as follows: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
- FIG 4
Promoter occupancy of Cap2 requires the bZIP, HAP4L, and C-terminal conserved domains. (A and B) The deletion of either ΔC1, ΔC2, ΔC3, and ΔC4 or mutation in bZIP domain (cap2-1) or deletion of HAP4L domain (cap2-2) abolished Cap2 recruitment at ACO1 (A) and CYC1 (B) promoters compared to the wild-type control under iron-depleted conditions. (C) Cap2 is not recruited to the FRP1 promoter in the WT and mutants. The values that are significantly different are indicated by bars and asterisks as follows: **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
