PAH1-encoded phosphatidate phosphatase plays a role in the growth phase- and inositol-mediated regulation of lipid synthesis in Saccharomyces cerevisiae

Abstract

In the yeast Saccharomyces cerevisiae, the synthesis of phospholipids in the exponential phase of growth occurs at the expense of the storage lipid triacylglycerol. As exponential phase cells progress into the stationary phase, the synthesis of triacylglycerol occurs at the expense of phospholipids. Early work indicates a role of the phosphatidate phosphatase (PAP) in this metabolism; the enzyme produces the diacylglycerol needed for the synthesis of triacylglycerol and simultaneously controls the level of phosphatidate for the synthesis of phospholipids. Four genes (APP1, DPP1, LPP1, and PAH1) encode PAP activity in yeast, and it has been unclear which gene is responsible for the synthesis of triacylglycerol throughout growth. An analysis of lipid synthesis and composition, as well as PAP activity in various PAP mutant strains, showed the essential role of PAH1 in triacylglycerol synthesis throughout growth. Pah1p is a phosphorylated enzyme whose in vivo function is dependent on its dephosphorylation by the Nem1p-Spo7p protein phosphatase complex. nem1Δ mutant cells exhibited defects in triacylglycerol synthesis and lipid metabolism that mirrored those imparted by the pah1Δ mutation, substantiating the importance of Pah1p dephosphorylation throughout growth. An analysis of cells bearing PPAH1-lacZ and PPAH1-DPP1 reporter genes showed that PAH1 expression was induced throughout growth and that the induction in the stationary phase was stimulated by inositol supplementation. A mutant analysis indicated that the Ino2p/Ino4p/Opi1p regulatory circuit and transcription factors Gis1p and Rph1p mediated this regulation.

Keywords: Diacylglycerol; Lipids; Phosphatase; Phosphatidate; Phospholipid; Phospholipid Metabolism; Triacylglycerol; Yeast.

FIGURE 1.

Effect of the pah1Δ and nem1Δ mutations on the composition of neutral lipids and total phospholipids during growth. Wild type, pah1Δ, and nem1Δ mutant cells were grown in SC medium in the presence of [2-¹⁴C]acetate (1 μCi/ml). At the indicated time intervals, lipids were extracted, separated by TLC, visualized by phosphorimaging analysis, and their relative amounts were analyzed with ImageQuant software. The percentage of each lipid was normalized to the total ¹⁴C-labeled chloroform fraction. Each data point represents the average of three independent experiments ± S.D. (error bars). Error bars are hidden behind some symbols. PL, phospholipids; FA, fatty acids; Erg, ergosterol; ErgE, ergosterol esters.

FIGURE 2.

Effect of the pah1Δ and nem1Δ mutations on the synthesis of neutral lipids and total phospholipids during growth. Wild type, pah1Δ, and nem1Δ mutant cells were grown in SC medium to the indicated time intervals. Cells were then harvested, normalized to equal cell densities, and incubated with [2-¹⁴C]acetate (5.0 μCi/ml) for 20 min. Lipids were extracted, separated by TLC, and visualized by phosphorimaging analysis, and their relative amounts were analyzed with ImageQuant software. The percentage of each lipid was normalized to the total ¹⁴C-labeled chloroform fraction. Each data point represents the average of three independent experiments ± S.D. (error bars). Error bars are hidden behind some symbols. PL, phospholipids; FA, fatty acids; Erg, ergosterol; ErgE, ergosterol esters.

FIGURE 3.

Growth phase-mediated regulation of PAH1-encoded PAP activity. Wild type, dpp1Δ lpp1Δ, app1Δ dpp1Δ lpp1Δ, and pah1Δ dpp1Δ lpp1Δ mutant cells were grown in SC medium to the indicated time intervals. A, cell densities were monitored with a spectrophotometer at 600 nm. B, cell extracts were prepared and used for the assay of PAP activity. PAP activity from cells harvested at the indicated time of growth is shown. Each data point represents the average of triplicate enzyme determinations from two independent experiments ± S.D. (error bars). C, PAP activity was measured in the cytosol and total membrane fractions of dpp1Δ lpp1Δ mutant cells. Each data point represents the average of triplicate absorbance determinations ± S.D. (error bars). Error bars are hidden behind some symbols.

FIGURE 4.

Growth phase-mediated regulation of P_PAH1-lacZ reporter gene expression. A, wild type cells bearing the P_PAH1-lacZ reporter gene were grown from the exponential to stationary phases in SC medium. B, stationary phase cells bearing the P_PAH1-lacZ reporter gene were harvested and resuspended in fresh SC medium to resume vegetative growth. At the indicated time intervals, cell extracts were prepared and assayed for β-galactosidase activity. Each data point represents the average of triplicate enzyme determinations from two independent experiments ± S.D. (error bars). Error bars are hidden behind some symbols.

FIGURE 5.

Growth phase-mediated regulation of P_PAH1-DPP1 reporter gene expression. dpp1Δ mutant cells bearing plasmids pRS415 (vector), pGH339 (P_PAH1-DPP1), or pGH201 (P_DPP1-DPP1) were grown in SC medium. At the indicated time intervals, cell extracts were prepared and subjected to immunoblot analysis using anti-Dpp1p, anti-Pah1p, or anti-Pgk1p (loading control) antibodies. Portions of representative blots of two experiments are shown in the figure. The positions of the lanes from each blot were aligned with Adobe Photoshop.

FIGURE 6.

Effect of inositol on the growth phase-mediated regulation of P_PAH1-lacZ reporter gene expression. Wild type cells bearing the P_PAH1-lacZ reporter gene were grown in the absence or presence of the indicated concentrations of inositol. Cells were harvested in the exponential and stationary phases of growth; cell extracts were prepared and assayed for β-galactosidase activity. Each data point represents the average of triplicate enzyme determinations from two independent experiments ± S.D. (error bars). Error bars are hidden behind some symbols.

FIGURE 7.

Expression of P_PAH1-lacZ reporter genes with promoter deletions in exponential and stationary phase cells grown in the absence or presence of inositol. Wild type cells bearing the indicated P_PAH1-lacZ promoter genes were grown in the absence or presence of 75 μm inositol. Cells were harvested in exponential and stationary phases of growth; cell extracts were prepared and assayed for β-galactosidase activity. Each data point represents the average of triplicate enzyme determinations from a minimum of two independent experiments ± S.D. (error bars). The full-length promoter construct contains 1,000 bases upstream of the ATG start site. The number of bases in each reporter gene construct is denoted at the left side of the figure. The putative Gis1p, Rph1p, and Ino2p/Ino4p transcription factor-binding sites in the PAH1 promoter are indicated.

FIGURE 8.

Effects of the ino2Δ, ino4Δ, and opi1Δ mutations on the regulation of P_PAH1-lacZ reporter gene expression in exponential and stationary phase cells grown in the absence or presence of inositol. Wild type, ino2Δ, ino4Δ, and opi1Δ mutant cells bearing the P_PAH1-lacZ reporter gene were grown in the absence or presence of 75 μm inositol. Because of their inositol auxotrophy (83), the inositol-free medium used for the growth of the ino2Δ and ino4Δ mutants was supplemented with 10 μm inositol (142). Cells were harvested in exponential (E) and stationary (S) phases of growth; cell extracts were prepared and assayed for β-galactosidase activity. Each data point represents the average of triplicate enzyme determinations from a minimum of two independent experiments ± S.D. (error bars).

FIGURE 9.

Effects of the gis1Δ and rph1Δ mutations on the regulation of P_PAH1-lacZ reporter gene expression in exponential and stationary phase cells grown in the absence or presence of inositol. Wild type, gis1Δ, and rph1Δ mutant cells bearing the P_PAH1-lacZ reporter gene were grown in the absence or presence of 75 μm inositol. Cells were harvested in exponential and stationary phases of growth; cell extracts were prepared and assayed for β-galactosidase activity. Each data point represents the average of triplicate enzyme determinations from a minimum of two independent experiments ± S.D. (error bars).