Positive feedback regulates switching of phosphate transporters in S. cerevisiae

Abstract

The regulation of transporters by nutrient-responsive signaling pathways allows cells to tailor nutrient uptake to environmental conditions. We investigated the role of feedback generated by transporter regulation in the budding yeast phosphate-responsive signal transduction (PHO) pathway. Cells starved for phosphate activate feedback loops that regulate high- and low-affinity phosphate transport. We determined that positive feedback is generated by PHO pathway-dependent upregulation of Spl2, a negative regulator of low-affinity phosphate uptake. The interplay of positive and negative feedback loops leads to bistability in phosphate transporter usage--individual cells express predominantly either low- or high-affinity transporters, both of which can yield similar phosphate uptake capacity. Cells lacking the high-affinity transporter, and associated negative feedback, exhibit phenotypes that arise from hysteresis due to unopposed positive feedback. In wild-type cells, population heterogeneity generated by feedback loops may provide a strategy for anticipating changes in environmental phosphate levels.

Figure 1

Phosphate uptake and growth of wild-type and mutant strains. (A) EY57 (wild-type), EY105 (pho84Δ) and EY329 (pho84Δpho4Δ) were grown in logarithmic phase for 24–48 hours in high phosphate medium (SD medium containing 15 mM phosphate), transferred to SD or SD medium lacking phosphate for 4 hours at 30°C, and ³²P uptake measurements were performed as described previously (Wykoff and O’Shea, 2001). The initial uptake velocity of wild-type cells grown in no phosphate conditions is not displayed; the uptake velocity values approach 20 nmol phosphate min⁻¹ OD₆₀₀⁻¹. EY105 exhibited a 50% defect in phosphate uptake when inoculated from a plate and grown for 8–10 hours in SD medium (Wykoff and O’Shea, 2001), but this defect was suppressible by growth for 24–48 hours in SD medium containing 15 mM phosphate. (B) Strains EY57, EY105, EY152 (pho84Δpho81Δ), EY334 (pho84Δpho81Δpho80Δ), EY329, and EY1880 (pho84Δpho81Δ PHO4^SA1-4PA6) were grown overnight in SD medium, diluted to OD₆₀₀~0.3 and plated in 10-fold dilutions. The first three panels of photographs are of plates that were incubated for two days at 30°C. The last panel is a photograph of a plate that was incubated for 5 days at 30°C. In the second panel, the plate was overlaid with a substrate to detect Pho5 acid phosphatase activity (Bun-Ya et al., 1991).

Figure 2

Spl2 is necessary and overexpression is sufficient to down-regulate low-affinity phosphate transport. (A) Strains EY57 (wild-type), EY105 (pho84Δ), EY329 (pho84Δpho4Δ), and EY1718 (pho84Δ spl2Δ) were grown and plated as described in Figure 1, except that the YEPD + 8 mM AsO₄ plate was incubated for 3.5 days at 30°C. The aggregate V_max and K_m was calculated from phosphate uptake data of three independently grown cultures and the error is the standard deviation. Because there is negligible phosphate uptake in the pho84Δ strain, the K_m was not determined. (B) Strains EY57 (wild-type), EY1982 (pho87Δpho90Δpho91Δ), and EY1959 (PGK1pr-SPL2) were grown in high or no phosphate medium for 4 hours. Phosphate uptake measurements were performed and the aggregate kinetic constants were derived. The error is the standard deviation of three independent experiments. V_max and K_m values are reported in units of nmol P_i min⁻¹ OD600⁻¹ and μM P_i, respectively (C) Strains containing integrated versions of the PHO84 and PHO5 promoters controlling GFP expression (the PHO84pr-GFP reporter is integrated at the URA3 locus so that the wild-type copy of PHO84 is intact) were grown in high and no phosphate medium and assayed by flow cytometry. The fluorescence was background subtracted and normalized to total induction of wild-type cells. The errors are the standard deviation between the mean fluorescence of three independent cultures. The strains are EY2094 (PHO5pr-GFP), EY1981 (pho87Δpho90Δpho91ΔPHO84pr-GFP), EY1958 (PGK1pr-SPL2 PHO84pr-GFP), EY1995 (PHO84pr-GFP), EY2044 (pho87Δpho90Δpho91ΔPHO5pr-GFP), and EY2045 (PGK1pr-SPL2 PHO5pr-GFP).

Figure 3

Positive feedback through induction of Spl2 generates complex population dynamics. (A) Phosphate titration contour maps demonstrate the bistable response of wild-type cells in intermediate phosphate conditions. Strain EY1995 expressing PHO84pr-GFP was grown in no phosphate medium supplemented with the indicated concentrations of inorganic phosphate. Cells were grown at low density (10⁴–10⁵ cells per ml) to prevent phosphate depletion. After 18 hours of growth (at which time the levels of GFP expression had reached steady-state), cells were harvested and subjected to flow cytometry. Titration contour plots were generated for each phosphate concentration condition by normalizing to maximum peak height and creating a color map for all concentration measurements. (B) Phosphate uptake assays were conducted on subpopulations of wild-type EY2095 (PHO84pr-GFP) cells grown in intermediate phosphate (150 μM phosphate) and sorted into populations expressing high and low levels of GFP by flow cytometry. The K_m values of the two populations suggest that low-affinity phosphate transporters dominate the un-induced population and high-affinity transporters predominate in the induced population. (C) Strain EY2096 (PHO84pr-GFP spl2Δ) was grown and analyzed as in (A) and a contour map was generated from flow cytometry data.

Figure 4

Phenotypic switching of the complement of phosphate transporters is mediated by counteracting positive and negative feedback loops. Pho4, which is regulated by internal phosphate concentrations, controls the transcription of PHO84 and SPL2. Pho84, because of its ability to transport inorganic phosphate, serves as a negative feedback element in the network. Spl2 is a positive feedback element because it down-regulates the activity low-affinity phosphate transporters, reducing internal phosphate levels and leading to further activation of Pho4.