Fig 5.
Hac1 supports FLO11 expression during amino acid starvation. (A) Expression of FLO11::lacZ was assayed in the haploid Σ1278b wild-type yeast strain (WT) (RH3406) as well as in Δhac1 (RH3360), Δgcn4 (RH3407), and Δgcn4 Δhac1 (RH3408) mutant strains each carrying a chromosomally integrated FLO11::lacZ reporter. Cultures were grown to log phase in YNB in the absence (light gray bars, YNB) or presence of 10 mM 3AT (dark gray bars, +3AT) before specific β-galactosidase activities were measured. Relative FLO11 mRNA abundances determined by qRT-PCR were measured in haploid Σ1278b wild-type (WT) (RH2816), Δhac1 (RH3351), Δgcn4 (RH2676), and Δgcn4 Δhac1 (RH3402) yeast strains and normalized against CDC28. Experiments were performed from three independent cultures for each strain and condition, and cultivation was accomplished as described for FLO11::lacZ expression. The ΔCT method including efficiencies was used for quantification. Standard deviations are indicated as error bars. (B) The untransformed yeast strains described in panel A as well as a Δflo11 (RH2681) mutant strain were streaked out on solid YNB medium (nonstarved cells) and with 10 mM 3AT (histidine-starved cells), respectively. After incubation for 3 days at 30°C, adhesive growth was determined. Plates were photographed before (before wash) and after washing under a stream of water (after wash) to document remaining cells on the agar surface. The same yeast strains were grown in liquid YNB medium until reaching an optical density of 0.6 before 300 μl of each culture was transferred in a microtiter well. Cells were grown in the absence or presence of 5 or 10 mM 3AT to induce starvation-dependent adhesive growth. After incubation for 24 h at 30°C, sedimented cells were dyed with crystal violet and carefully washed. Finally, plates were photographed to document adhesive growth. (C) FLO11::lacZ expression was also determined in the diploid Σ1278b wild-type yeast strain (WT) (RH3417) as well as in diploid homo- and heterozygous Δhac1/Δhac1 (RH3362), Δhac1/HAC1 (RH3418), Δgcn4/Δgcn4 (RH2695), Δgcn4/GCN4 (RH3419), Δgcn4/Δgcn4 Δhac1/Δhac1 (RH3349), and Δgcn4/GCN4 Δhac1/HAC1 (RH3420) mutant yeast strains each carrying a chromosomally integrated FLO11::lacZ reporter. For testing amino acid starvation-induced adhesive growth, the diploid wild-type yeast strain (WT) (RH2656) as well as Δflo11/Δflo11 (RH2661), Δhac1/Δhac1 (RH3412), Δhac1/HAC1 (RH3413), Δgcn4/Δgcn4 (RH2658), Δgcn4/GCN4 (RH3414), Δgcn4/Δgcn4 Δhac1/Δhac1 (RH3415), and Δgcn4/GCN4 Δhac1/HAC1 (RH3416) mutant strains was used, and the assay was performed as described for panel B.