Regulation of the yeast triacylglycerol lipase TGl3p by formation of nonpolar lipids

Abstract

Tgl3p, the major triacylglycerol lipase of the yeast Saccharomyces cerevisiae, is a component of lipid droplets but is also present in the endoplasmic reticulum in a minor amount. Recently, it was shown that this enzyme can also serve as a lysophospholipid acyltransferase (Rajakumari, S., and Daum, G. (2010) Mol. Biol. Cell 21, 501-510). Here, we describe the effects of the presence/absence of triacylglycerols and lipid droplets on the functionality of Tgl3p. In a dga1Δlro1Δare1Δare2Δ quadruple mutant lacking all four triacylglycerol- and steryl ester-synthesizing acyltransferases and consequently the lipid droplets, the gene expression of TGL3 was only slightly altered. In contrast, protein level and stability of Tgl3p were markedly reduced in the absence of lipid droplets. Under these conditions, the enzyme was localized to the endoplasmic reticulum. Even the lack of the substrate, triacylglycerol, affected stability and localization of Tgl3p to some extent. Interestingly, Tgl3p present in the endoplasmic reticulum seems to lack lipolytic as well as acyltransferase activity as shown by enzymatic analysis and lipid profiling. Thus, we propose that the activity of Tgl3p is restricted to lipid droplets, whereas the endoplasmic reticulum may serve as a parking lot for this enzyme.

Keywords: Acyltransferase; Endoplasmic Reticulum (ER); Lipase; Lipid Droplets; Triacylglycerol; Yeast.

FIGURE 1.

Gene expression, protein level, and stability of Tgl3p in the absence of LD. A, relative gene expression of TGL3 in wild type (WT) (black bar) and QM (gray bar) measured by RT-PCR. Wild type was set at 1. Data are mean values from three independent experiments with the respective deviation. B, protein analysis of Tgl3-Myc of total cell extracts from wild type and QM grown to the stationary phase. C, relative protein level of Tgl3-Myc of total cell extracts from wild type (black bar) and QM (gray bar) obtained by three Western blots was calculated using ImageJ program. D, Western blot analysis of Tgl3-Myc was performed with total cell extracts from wild type and QM grown for time periods as indicated after addition of 100 μg/ml cycloheximide to cells grown to mid-logarithmic phase. GAPDH was used as loading control. E, relative protein stability in wild type and QM obtained by three Western blots was calculated using the ImageJ program. Protein half-life is shown. Western blot analyses are representative of at least two independent experiments. RQ, relative quantity.

FIGURE 2.

Localization and lipase activity of Tgl3p in the absence of LD. A, Western blot analysis of Tgl3-Myc in homogenate (Hom), 30,000 × g microsomes (M30), 40,000 × g microsomes (M40), cytosol (Cyt), and LD fraction (LD) from wild type (WT) and QM grown to the stationary phase. Primary antibodies were directed against the Myc tag, Wbp1p (ER marker), and GAPDH (cytosolic marker). Western blot analyses are representative of at least two independent experiments. B, fluorescence microscopy of PGal1-GFP-Tgl3 in wild type and QM grown to late logarithmic phase after induction with galactose for 4 h. Two different sections from the QM strain are shown. Size bar, 5 μm. C, analysis of TG lipase activity of LD and 30,000 × g ER fractions from wild type and QM overexpressing TGL3. Experiments were performed in triplicates and are representative of at least two independent experiments. Data are mean values with the respective deviation. DIC, differential interference contrast.

FIGURE 3.

Localization of Tgl3p in yeast strains lacking nonpolar lipid-synthesizing enzymes. A, Western blot analysis of Tgl3-Myc in homogenate and LD fractions from wild type (WT), lro1Δare1Δare2Δ, and dga1Δlro1Δ grown to the stationary phase. B, relative protein levels of Tgl3-Myc from total cell extracts of wild type (black bar), lro1Δare1Δare2Δ (gray bar), and dga1Δlro1 (light gray bar) obtained by three Western blots were calculated using ImageJ program. C, Western blot analysis of Tgl3-Myc was performed with total cell extracts from lro1Δare1ΔareΔ and dga1Δlro1Δ grown for time periods as indicated after addition of 100 μg/ml cycloheximide to cells grown to the mid-logarithmic phase. D, relative protein stability in lro1Δare1ΔareΔ and dga1Δlro1Δ obtained by three Western blots was calculated using ImageJ program. Protein half-life is shown. E, Western blot analysis of Tgl3-Myc in homogenate (Hom), 30,000 × g microsomes (M30), 40,000 × g microsomes (M40), and cytosol (Cyt). Primary antibodies were directed against the Myc tag, Wbp1p (ER marker), GAPDH (cytosolic marker), Ayr1p (LD marker), and Erg1p (LD marker). Western blot analyses are representative of at least two independent experiments. RQ, relative quantity.

FIGURE 4.

Gene expression, protein level, and localization of Tgl3p in different mutants. A, relative gene expression of TGL3^WT, tgl3^S237A, tgl3^H298A, and tgl3^S237A/H298A was measured by RT-PCR. TGL3^WT was set at 1. Data are mean values from three independent experiments with the respective deviations. B, protein analysis of Tgl3^WT, Tgl3^S237A, Tgl3^H298A, and Tgl3^S237A/H298A from total cell extracts grown to stationary phase. GAPDH was used as loading control. C, Western blot analysis of Tgl3^WT, Tgl3^S237A, Tgl3^H298A, and Tgl3^S237A/H298A in homogenate (Hom) and LD fractions from cells grown to the stationary phase. D, Western blot analysis of Tgl3p and variants in homogenate (Hom), 30,000 × g microsomes (M30), and 40,000 × g microsomes (M40). Primary antibodies were directed against the HA tag, Wbp1p (ER marker), GAPDH (cytosolic marker), Ayr1p (LD marker), and Erg6p (LD marker). Western blot analyses are representative of at least two independent experiments. RQ, relative quantity.

FIGURE 5.

Lipid analysis of yeast strains lacking major nonpolar lipid-synthesizing enzymes. A, relative amounts of total phospholipids/mg of protein of total cell extracts from wild type (WT) and mutants with additional deletion of TGL3 grown to the stationary phase. Data are mean values of three independent experiments with respective deviations. The wild type (45 mg ± 4.9 mg of phospholipids/mg of protein) was set at 100%. B, analysis of lysophosphatidylethanolamine acyltransferase activity in 30,000 × g ER fractions from QM and QMtgl3Δ. Assays were performed in triplicate from at least two independent biological samples. Data are mean values with the respective deviation. C, relative amount of DG from wild type and QM with additional deletion of TGL3 in cells grown to the stationary phase. Data are mean values of three independent experiments with respective mean deviations. Wild type was set at 100%.