Fig 1.
Transcriptional regulation of the PHO regulon in high and low Pi conditions.
Fig 2.
Identification of mutants with altered PHO84 expression in low and high Pi conditions.
(A) Generation of single mutants harboring the PHO84 reporter with the SGA method. The PHO84 reporter consists of PHO84 promoter-driven Venus and TEF2 promoter-driven mCherry. Each single mutant in the library denoted by xxxΔ is kanamycin (G418)-resistant. (B) The distributions of the PHO84 reporter levels in single cells in the pho81Δ and pho80Δ strains. Log2 intensity ratio of Venus to mCherry (log2(YFP/RFP)) was used to quantify the PHO84 expression level. (C, D) The PHO84 reporter levels of single mutants in the library measured in 50 μM Pi and 1 mM Pi conditions. The PHO84 reporter level of each mutant was normalized to that of the wild type value in each Pi concentration (Materials and methods). Red dashed lines in (C) and (D) indicate the PHO84 reporter levels with p-values less than 0.001 estimating the maximum range of the PHO84 reporter levels that the wild type exhibits in each Pi concentration. The mutants in black are previously identified mutants and the one in red is identified in this study.
Fig 3.
Identification of genes acting upstream of the Pho80/Pho85/Pho81 kinase complex.
(A) A schematic diagram depicting the expected outcome of epistasis analysis depending on whether or not a mutant is defective in the signaling process upstream of Pho80/Pho85. Genes in red act upstream of Pho80/Pho85 and those in blue do not act upstream of Pho80/Pho85. (B) The PHO84 reporter levels of double mutants carrying the less induced hits and pho80DΔ in 50 μM Pi conditions. All 380 less induced hits were used to generate the double mutants. The PHO84 reporter levels of double mutants in (B) were normalized to that of pho80DΔ. A red dashed line in (B) indicates the maximum PHO84 reporter level of double mutants generated by one of the known downstream genes (pho80DΔ gcn5Δ). (C) The PHO84 reporter levels of double mutants carrying the less repressed hits and pho81Δ in 1 mM Pi conditions. All 243 less repressed hits were used to generate the double mutants. The PHO84 reporter levels of double mutants in (C) were normalized to that of pho81Δ. In (B) and (C), mutants in blue and red are defective in signaling process downstream and upstream of Pho80/Pho85, respectively. (D) A schematic diagram depicting adenine nucleotide metabolism. A gene in red is identified in this study and those in bold black are previously identified.
Fig 4.
Adenine nucleotide levels in the wild type in no Pi and in adk1Δ, aah1Δ and ado1Δ in high Pi.
(A) [ATP], [ADP], and [AMP] in wild type (WT) cells over time grown in no Pi medium. All the adenine nucleotide concentrations at each time point were normalized to those in 10 mM Pi. Note that the PHO pathway in no Pi is activated within 15 minutes. Adenine nucleotide levels at each time point were measured three times and the error bars in (A) are standard errors. (B) [ATP], [ADP], and [AMP] in WT, adk1Δ, aah1Δ, and ado1Δ in 10 mM Pi. Adenine nucleotide levels in the three mutants were measured two times and the error bars in (B) are standard errors.
Fig 5.
The PHO pathway in vip1Δ mutant is inducible in 50 uM Pi, but its induction kinetics are slower than the wild type.
(A) The PHO84 reporter levels of the wild type over time in 50 uM Pi. (B) The PHO84 reporter levels of vip1Δ over time in 50 uM Pi. Time 0 data in (A) and (B) were obtained in 10 mM Pi before cells were inoculated into 50 μM Pi.
Fig 6.
VIP1 is required for constitutive activation of the PHO pathway in ado1Δ and aah1Δ.
The PHO84 reporter levels in all the strains in Fig 6 were averaged over 3 measurements and normalized to the PHO84 reporter level in the wild type in 1 mM Pi conditions. Error bars represent the standard deviation of the normalized PHO84 reporter levels in the mutants.