Yeh1 constitutes the major steryl ester hydrolase under heme-deficient conditions in Saccharomyces cerevisiae

Abstract

Steryl esters are stored in intracellular lipid droplets from which they are mobilized upon demand and hydrolyzed to yield free sterols and fatty acids. The mechanisms that control steryl ester mobilization are not well understood. We have previously identified a family of three lipases of Saccharomyces cerevisiae that are required for efficient steryl ester hydrolysis, Yeh1, Yeh2, and Tgl1 (R. Köffel, R. Tiwari, L. Falquet, and R. Schneiter, Mol. Cell. Biol. 25:1655-1668, 2005). Both Yeh1 and Tgl1 localize to lipid droplets, whereas Yeh2 is localized to the plasma membrane. To characterize the precise function of these three partially redundant lipases, we examined steryl ester mobilization under heme-deficient conditions. S. cerevisiae is a facultative anaerobic organism that becomes auxotrophic for sterols and unsaturated fatty acids in the absence of molecular oxygen. Anaerobic conditions can be mimicked in cells that are deficient for heme synthesis. We here report that Yeh1 is the sole active steryl ester hydrolase under such heme-deficient conditions, indicating that Yeh1 is activated whereas Yeh2 and Tgl1 are inactivated by the lack of heme. The heme-dependent activation of Yeh1 is mediated at least in part by an increase in steady-state levels of Yeh1 at the expense of Yeh2 and Tgl1 in exponentially growing cells. This increase in steady-state levels of Yeh1 requires Rox3, a component of the mediator complex that regulates transcription by RNA polymerase II. These data thus provide the first link between fat degradation and the transcriptional control of lipase activity in yeast.

FIG. 1.

YEH1 is required for steryl ester mobilization in a heme-deficient background. (A) Heme-deficient wild-type (YRS1707) and yeh1Δ mutant (YRS1710) cells were labeled for 16 h with [¹⁴C]cholesterol, and the kinetics of steryl ester mobilization in vivo was analyzed by determining steryl ester levels at 0, 2, 4, and 6 h after the dilution of cells into fresh media. Lipids were extracted and analyzed by TLC, as described in Materials and Methods. Chol, cholesterol; CE, cholesteryl esters. (B) Levels of [¹⁴C]cholesteryl esters were quantified by radioscanning of TLC plates and set in relation to the levels at time zero (100%). Values represent means and standard deviations from two independent experiments.

FIG. 2.

YEH2 and TGL1 do not contribute to steryl ester hydrolysis under heme-deficient conditions. Heme-deficient lipase triple (yeh1Δ yeh2Δ tgl1Δ, YRS1922) and double (yeh1Δ tgl1Δ, YRS1923; yeh2Δ tgl1Δ, YRS1961; and yeh1Δ yeh2Δ, YRS2045) mutant cells were labeled for 16 h with [¹⁴C]cholesterol, and the kinetics of steryl ester mobilization in vivo was analyzed after the dilution of cells into fresh media. Lipids were extracted and analyzed by TLC, as described in Materials and Methods. Levels of free and esterified [¹⁴C]cholesterol were quantified by radioscanning of TLC plates. Data shown are representative of two independent experiments, with standard deviations of less than 5% between experiments.

FIG. 3.

Yeh1 is active against different steryl ester substrates. Heme-deficient wild-type (YRS1707), lipase triple mutant (YRS1922), and lipase double mutant (YRS1923, YRS1961, and YRS2045) cells were precultivated in media containing ergosterol, cholesterol, or lanosterol, and the steryl ester pool was labeled by incubating cells with [³H]palmitic acid for 16 h. Cells were then diluted into fresh media containing the same sterol as that used for the precultivation, and samples were removed after 0 h and 6 h of growth. Lipids were extracted and analyzed by TLC, and levels of [³H]palmitate in cholesteryl, lanosteryl, and ergosteryl esters were quantified by radioscanning. Values represent means and standard deviations from two independent experiments.

FIG. 4.

Heme deficiency results in increased steady-state levels of Yeh1 at the expense of Yeh2 and Tgl1. (A) Heme-deficient cells expressing a GFP-tagged version of Yeh1 (YRS2046), Yeh2 (YRS2048), and Tgl1 (YRS2047) were cultivated in media containing either ALA or cholesterol (Chol) plus Tween 80 for 16 h, and the subcellular localization of the lipases was examined by fluorescence microscopy. Bar, 5 μm. (B) Heme-deficient cells expressing GFP-tagged lipases were precultivated in media containing either ALA or cholesterol plus Tween 80 for 16 h and diluted to an OD₆₀₀ of 0.8, and samples were removed at the indicated time points. Steady-state levels of the GFP-tagged enzymes were analyzed by Western blotting, using Wbp1 as a loading control. (C) Signal intensities on Western blots were quantified by densitometry. Data represent means ± standard errors of the means (n = 3). Significance of the difference between the steady-state levels of Yeh1-GFP and Tgl1-GFP and between Yeh1-GFP and Yeh2-GFP, as based on a two-tailed unpaired t test, is indicated by asterisks (*, P < 0.05; **, P < 0.01). (D) Northern analysis of transcript levels. Heme-deficient wild-type cells (YRS1707) were cultivated in media containing either ALA or cholesterol plus Tween 80 for 16 h and diluted to an OD₆₀₀ of 0.8, and samples were removed at the indicated time points. RNA was extracted, and transcript levels of YEH1, TGL1, and actin (ACT1) were determined by Northern blotting.

FIG. 5.

The heme-dependent inactivation of Yeh2 and Tgl1 is overcome by overexpression of the enzymes. (A) Heme-deficient wild-type cells (YRS1707), yeh1Δ yeh2Δ tgl1Δ triple mutant cells (YRS1922), and cells expressing an N-terminally GFP-tagged version of Yeh1 (YRS2184), Yeh2 (YRS2183), and Tgl1 (YRS2182) from a regulatable GAL1 promoter were labeled for 16 h with [¹⁴C]cholesterol, and the kinetics of steryl ester mobilization was analyzed after dilution of cells into fresh media containing either glucose or galactose. Cells were cultivated for 6 h, lipids were extracted and analyzed by TLC, and the relative content of [¹⁴C]cholesterol in the steryl ester pool was quantified by radioscanning of TLC plates. Values represent means and standard deviations from two independent experiments. (B) The level of repression or induction of the GFP-tagged lipases under the experimental conditions used for panel A was monitored by Western blot analysis, using Kar2 as a loading control.

FIG. 6.

ROX3 is required for efficient mobilization of steryl esters under heme deficiency. (A) Heme-deficient wild-type (YRS1707), lipase triple mutant (YRS1922), and rox3Δ mutant (YRS1766) cells were labeled for 16 h with [¹⁴C]cholesterol, and the kinetics of steryl ester mobilization in vivo was analyzed by determining steryl ester levels at 0 and 6 h after the dilution of cells into fresh media. Lipids were extracted and analyzed by TLC, and the relative content of [¹⁴C]cholesterol in the steryl ester pool was quantified by radioscanning of TLC plates. Values represent means and standard deviations from two independent experiments. (B) ROX3 is required for induction of Yeh1-GFP in lipid-supplemented media. Heme-deficient wild-type cells (YRS2046) and rox3Δ mutant cells expressing Yeh1-GFP (YRS2740) were cultivated for 24 h in media containing either ALA or cholesterol plus Tween 80, and levels of Yeh1-GFP were determined by quantification of Western blots, using Wbp1 as a loading control. Signal intensities were quantified by densitometry. Data represent means ± standard errors of the means (n = 4). Significance of the difference between the steady-state levels of Yeh1-GFP in wild-type and rox3Δ mutant cells, as based on a two-tailed unpaired t test, is indicated by asterisks (P = 0.0042).