Sterol homeostasis in eukaryotic cells relies on the reciprocal interconversion of free sterols and steryl esters. The formation of steryl esters is well characterized, but the mechanisms that control steryl ester mobilization upon cellular demand are less well understood. We have identified a family of three lipases of Saccharomyces cerevisiae that are required for efficient steryl ester mobilization. These lipases, encoded by YLL012/ YEH1, YLR020/YEH2, and TGL1, are paralogues of the mammalian acid lipase family, which is composed of the lysosomal acid lipase, the gastric lipase, and four novel as yet uncharacterized human open reading frames. Sterols are essential lipids of eukaryotic cells. They are present in two major forms: free sterols and steryl esters. Free sterols are synthesized in the endoplasmic reticulum (ER) membrane but are greatly enriched at the plasma membrane, which harbors 90% of the free-sterol pool of a cell (27). Steryl esters, on the other hand, serve as a storage form for fatty acids and sterols that are deposited in intracellular lipid bodies or particles. The conversion of free sterols and acyl coenzymes A to steryl esters is localized to the ER (14). The formation of steryl esters is important to maintain sterol homeostasis, as the steryl ester pool conceptually serves to buffer both excess and a lack of free sterols (12). The genes required for the synthesis of steryl esters in yeast have been identified, and mutants that lack steryl esters are viable, indicating that their synthesis is not essential under standard growth conditions (52, 53). The reverse process, however, how endogenously synthesized steryl esters are mobilized from their stores and hydrolyzed to release free sterols and fatty acids, is less well understood, even though cleavage of this ester bond is generally thought to be catalyzed by a lipase.Lipases constitute a heterogeneous family of proteins with carboxyl esterase activity and are activated by a lipid-water interface (for reviews, see references 34 and 51). Crystallographic analysis revealed that lipases are remarkably similar in structure despite low overall sequence conservation. They belong to the alpha/beta hydrolase fold family of enzymes with diverse hydrolytic functions. Their catalytic domain consist of a predominantly parallel beta-sheet structure of eight beta sheets connected by helical loops of various lengths that contain the catalytic-triad residues serine, aspartic acid, and histidine (10,37,42). The nucleophilic serine of this triad is itself part of the nearly ubiquitous lipase consensus sequence motif GXSXG (17). These three amino acids, assisted by the dipolar oxyanion hole, which stabilizes the charge distribution of the transition state, catalyze the hydrolysis of the ester bond (37).Only a few gene products with lipolytic activity against neutral lipids have been characterized in Saccharomyces cerevisiae. Tgl1 has been proposed to be a triglyceride-specific lipase on the basis of its homology to lipases from humans and rats, but enzymat...

Sterol homeostasis in eukaryotic cells relies on the reciprocal interconversion of free sterols and steryl esters. Here we report the identification of a novel reversible sterol modification in yeast, the sterol acetylation/deacetylation cycle. Sterol acetylation requires the acetyltransferase ATF2, whereas deacetylation requires SAY1, a membrane-anchored deacetylase with a putative active site in the ER lumen. Lack of SAY1 results in the secretion of acetylated sterols into the culture medium, indicating that the substrate specificity of SAY1 determines whether acetylated sterols are secreted from the cells or whether they are deacetylated and retained. Consistent with this proposition, we find that acetylation and export of the steroid hormone precursor pregnenolone depends on its acetylation by ATF2, but is independent of SAY1-mediated deacetylation. Cells lacking Say1 or Atf2 are sensitive against the plant-derived allylbenzene eugenol and both Say1 and Atf2 affect pregnenolone toxicity, indicating that lipid acetylation acts as a detoxification pathway. The fact that homologues of SAY1 are present in the mammalian genome and functionally substitute for SAY1 in yeast indicates that part of this pathway has been evolutionarily conserved.