The Saccharomyces cerevisiae Lipin homolog is a Mg2+-dependent phosphatidate phosphatase enzyme

Abstract

Mg(2+)-dependent phosphatidate (PA) phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) catalyzes the dephosphorylation of PA to yield diacylglycerol and P(i). In this work, we identified the Saccharomyces cerevisiae PAH1 (previously known as SMP2) gene that encodes Mg(2+)-dependent PA phosphatase using amino acid sequence information derived from a purified preparation of the enzyme (Lin, Y.-P., and Carman, G. M. (1989) J. Biol. Chem. 264, 8641-8645). Overexpression of PAH1 in S. cerevisiae directed elevated levels of Mg(2+)-dependent PA phosphatase activity, whereas the pah1Delta mutation caused reduced levels of enzyme activity. Heterologous expression of PAH1 in Escherichia coli confirmed that Pah1p is a Mg(2+)-dependent PA phosphatase enzyme and showed that its enzymological properties were very similar to those of the enzyme purified from S. cerevisiae. The PAH1-encoded enzyme activity was associated with both the membrane and cytosolic fractions of the cell, and the membrane-bound form of the enzyme was salt-extractable. Lipid analysis showed that mutants lacking PAH1 accumulated PA and had reduced amounts of diacylglycerol and its derivative triacylglycerol.ThePAH1-encoded Mg(2+)-dependent PA phosphatase shows homology to mammalian lipin, a fat-regulating protein whose molecular function is unknown. Heterologous expression of human LPIN1 in E. coli showed that lipin 1 is also a Mg(2+)-dependent PA phosphatase enzyme.

FIGURE 1. Peptide sequences derived from purified Mg²⁺-dependent PA phosphatase (Pah1p)

A 91-kDa protein present in a partially purified preparation of Mg²⁺-dependent PA phosphatase was subjected to trypsin digestion followed by mass spectrometry analysis of 23 peptide fragments. The figure shows the positions and sequences of the non-overlapping peptides derived from the Mg²⁺-dependent PA phosphatase protein (Pah1p). The position of the HAD-like domain in Pah1p is also indicated in the figure.

FIGURE 2. The effects of the pah1Δmutation and PAH1 gene overexpression on Mg²⁺-dependent PA phosphatase activity in S. cerevisiae, and immunoblot analysis of Pah1p^HA

A, cytosolic and membrane fractions were prepared from the indicated cells at the exponential phase of growth and assayed for Mg²⁺-dependent PA phosphatase activity. The data shown were determined from triplicate enzyme determinations + S.D. B, a sample (15 μg) of cell extract derived from dpp1Δ lpp1Δ cells overexpressing the PAH1^HA gene were subjected to immunoblot analysis using anti-HA antibodies (1 μg/ml). The positions of Pah1p^HA and molecular mass standards are indicated in the figure.

FIGURE 3. Effect of NaCl on the membrane association of Mg²⁺-dependent PA phosphatase activity

Membranes from the indicated cells were suspended in buffer containing 0.5 M NaCl at a final protein concentration of 1 mg/ml. After incubation with shaking for 5 min at 4 °C, the suspensions were centrifuged at 100,000 x g for 1 h at 4 °C. The pellet fraction from each sample was suspended in the same volume of buffer as the supernatant fraction. Equal volumes (10 μl) of the fractions were assayed for Mg²⁺-dependent PA phosphatase activity. The data shown were determined from triplicate enzyme determinations + S.D.

FIGURE 4. Effect of NEM on the Mg²⁺-dependent PA phosphatase activity in S. cerevisiae wild type, dpp1Δ lpp1Δ, and pah1Δ dpp1Δ lpp1Δ cells

The cytosolic fraction from the indicated cells was assayed for Mg²⁺-dependent PA phosphatase activity in the absence and presence of 20 mM NEM. The data shown were determined from triplicate enzyme determinations + S.D.

FIGURE 5. SDS-PAGE of the PAH1 gene product purified from E. coli and protein concentration dependence of its Mg²⁺-dependent PA phosphatase activity

A, samples of molecular mass standards (Std), an E. coli cell extract, and the purified His-tagged PAH1-encoded protein were subjected to SDS-PAGE and stained with Coomassie Blue. The position of the recombinant PAH1-encoded protein (rPah1p) is indicated. B, the Mg²⁺-dependent PA phosphatase activity of the purified recombinant PAH1-encoded enzyme was measured with the indicated protein content.

FIGURE 6. Effect of pH, MgCl₂, Triton X-100, and PA surface concentration on PAH1-encoded Mg²⁺-dependent PA phosphatase activity

Purified recombinant PAH1-encoded enzyme was assayed for Mg²⁺-dependent PA phosphatase activity at the indicated pH values with 50 mM Tris-maleate-glycine buffer (A); the indicated concentrations of Triton X-100 (B); the indicated concentrations of MgCl₂ (C); and the indicated surface concentrations (mol %) of PA (D). The molar concentration of PA was held constant at 0.2 mM. The data shown were determined from triplicate enzyme determinations + S.D.

FIGURE 7. Effect of temperature on the growth of S. cerevisiae wild type, pah1Δ, pah1Δ dpp1Δ lpp1Δ, and dpp1Δ lpp1Δ cells

The indicated cells were grown to saturation in YEPD medium and diluted to a density of 2 x 10⁷ cells per ml at 30 °C. Serial dilutions (1:10) of the cells were spotted (5 μl) onto YEPD plates, and growth was scored after 2 days of incubation at 30 and 37 °C.

FIGURE 8. Phospholipid composition of the S. cerevisiae wild type, pah1Δ, pah1Δ dpp1Δ lpp1Δ, and dpp1Δ lpp1Δ cells in the exponential and stationary phases of growth

The indicated cells were grown to the exponential (A) and stationary (B) phases of growth in the presence of ³²P_i (10 μCi/ml). Phospholipids were extracted, separated by two-dimensional thin-layer chromatography, and the images were subjected to ImageQuant analysis. The percentages shown for the individual phospholipids were normalized to the total ³²P-labeled chloroform-soluble fraction that included sphingolipids and unidentified phospholipids. Each data point represents the average of three experiments ± S.D. Abbreviations: PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; PA, phosphatidate.

FIGURE 9. Neutral lipid composition of the S. cerevisiae wild type, pah1Δ, pah1Δ dpp1Δ lpp1Δ, and dpp1Δ lpp1Δ cells in the exponential and stationary phases of growth

The indicated cells were grown to the exponential (A) and stationary (B) phases of growth in the presence of [2-¹⁴C]acetate (1 μCi/ml). Lipids were extracted, separated by one-dimensional thin-layer chromatography, and the images were subjected to ImageQuant analysis. The percentages shown for the individual lipids were normalized to the total ¹⁴C-labeled chloroform-soluble fraction, which also contained phospholipids and unidentified neutral lipids. Each data point represents the average of three experiments ± S.D. Abbreviations: DAG, diacylglycerol; TAG, triacylglycerol; Erg, ergosterol; ErgE, ergosterol ester; FA, fatty acid.