As an important step toward understanding sterol homeostasis at the cellular level, the aim of this study was to identify the steryl ester-hydrolyzing enzyme(s) of yeast. By a candidate gene approach, we identified a family of potential lipases with a predicted alpha/beta hydrolase-associated lipase domain as being required for steryl ester hydrolysis in vivo and in vitro.
The three lipases encoded by YEH1
, and TGL1
constitute the yeast paralogues of the mammalian acid lipase family, which includes lysosomal acid lipase A (LIPA), the enteric gastric/lingual lipase LIPF, and four novel members that are predicted on the basis of the human genome sequence, LIPL1 to LIPL4 (3
). The lysosomal acid lipase is a key enzyme in the intracellular degradation of neutral lipids that have been internalized through receptor-mediated endocytosis of lipoprotein particles (19
). The enzyme hydrolyzes cholesteryl esters and triacylglycerols, releasing free cholesterol and fatty acids. The released cholesterol rapidly equilibrates with the cellular pool to regulate its endogenous synthesis, esterification, and receptor-mediated uptake (11
). Feedback regulation of sterol synthesis by the steryl ester pool in yeast is poorly characterized, but the lipase mutants now provide an important tool to address this pathway genetically. LIPA is active toward both steryl esters and triacylglycerols (5
); the yeast enzymes, on the other hand, appear to be more specific for steryl esters, as triacylglycerol mobilization in vivo is not affected in triple-mutant cells.
LIPA deficiency in humans causes two rare autosomal recessive disorders, Wolman disease and cholesterol ester storage disease (4
). Wolman disease is lethal within the first year of life because of hepatosplenomegaly, adrenal calcification, and massive accumulation of triglycerides and cholesteryl esters in these organs, as well as in macrophages and blood vessels. Cholesterol ester storage disease is a less severe disorder with longer survival, hepatomegaly, premature atherosclerosis, and dyslipoproteinemias. Patients with the milder form of this lipid storage disease retain some residual LIPA activity (39
). The crucial role of LIPA in steryl ester metabolism also prompted its consideration as an important factor in atherogenesis (6
LIPA localizes to endosomes, where it cleaves low-density-lipoprotein-derived substrates. Our subcellular localization of GFP-tagged versions of the three yeast lipases indicates that Yeh1 and Tgl1 both localize to lipid particles whereas Yeh2 is enriched in the plasma membrane. Lipid particle localization of two of the three yeast steryl ester hydrolases, Yeh1 and Tgl1, is likely to allow regulated access to their neutral lipid substrates, which are stored in these particles (29
). Intriguingly, however, both lipases appear to be membrane proteins on the basis of the detergent requirement for their solubilization. Yeh1 and Tgl1 are each proposed to be an integral membrane protein with an N-terminal transmembrane segment(s). This membrane anchoring is particularly remarkable in light of the fact that the lipid particle membrane has been proposed to be a monolayer rather than a normal bilayer membrane (29
). It would thus be interesting to examine how these transmembrane domains can be accommodated in a lipid monolayer. Alternatively, it might be conceivable that the two lipases localize to domains of the lipid particle membrane that retain a bilayer rather than a monolayer structure.
The role of Yeh2 in light of its plasma membrane localization is less obvious than that of lipid particle-localized Yeh1 and Tgl1. But given the fact that the active site of the enzyme is located in a luminal-extracellular compartment, it is tempting to speculate that Yeh2 may be important to cleave either extracellular or endocytosed steryl esters. While yeast readily takes up exogenous fatty acids, sterols are taken up only under anaerobic conditions (32
). The observation that Yeh2 alone in a yeh1
Δ double mutant does not efficiently mobilize steryl esters in vivo is consistent with a limiting access of the enzyme to the intracellular steryl ester pool, as predicted on the basis of the localization and topology of the enzyme. It is interesting that Yeh2 binds phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-3,4,5-triphosphate in vitro (54
). While phosphatidylinositol-3,4,5-triphosphate has not been detected in S. cerevisiae
), it remains to be seen whether these signaling lipids affect the activity of the enzyme. The size variants of Yeh2 detected on Western blots are likely to be due to O-linked glycosylation of the luminal domain of Yeh2, which would be consistent with the proposed type II topology of the enzyme.
Levels of steryl esters are coordinated with the growth phase (9
). It is thus necessary that their synthesis, mobilization, or both processes are tightly regulated. An interesting question for further studies will thus be to examine whether and, if so, how the lipases obtain regulated access to their substrates and to further investigate the molecular mechanism of this regulation. Mobilization of steryl esters from lipid particles requires energy and ongoing protein synthesis but is independent of microtubules (28
). Recent results obtained with mammalian cells suggest that an interplay between components that localize to the lipid droplets, such as perilipin and the lipase, in this case HSL, is important to coordinate substrate access. This interplay is regulated by protein kinase A to increase the rate of lipolysis by 30- to 100-fold (44
). Even though yeast lipid particles lack any obvious perilipin orthologue, it is interesting that both Yeh1 and Tgl1, but not Yeh2, contain potential cyclic-AMP-dependent protein kinase A phosphorylation sites. Taken together, the identification and topological characterization of three yeast lipases now provide the opportunity to use S. cerevisiae
to further characterize the molecular mechanisms that control sterol homeostasis at the cellular level.