Contribution of Are1p and Are2p to steryl ester synthesis in the yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, two acyl-CoA:sterol acyltransferases (ASATs) that catalyze the synthesis of steryl esters have been identified, namely Are2p (Sat1p) and Are1p (Sat2p). Deletion of either ARE1 or ARE2 ha s no effect on cell viability, and are1are2 double mutants grow in a simila r manner to wild-type despite the complete lack of cellular ASAT activity a nd steryl ester formation [Yang, H., Bard, M., Bruner, D. A., Gleeson, A., Deckelbaum, R. J., Aljinovic, G., Pohl, T. M., Rothstein, R. & Sturley, S. L. (1996) Science 272, 1353-1356; Yu, C., Kennedy, J., Chang, C. C. Y. & Ro thblatt, J. A. (1996) J. Biol. Chem. 271, 24157-24163]. Here we show that b oth Are2p and Are1p reside in the endoplasmic reticulum as demonstrated by measuring ASAT activity in subcellular fractions of are1 and are2 deletion strains. This localization was confirmed by fluorescence microscopy using h ybrid proteins of Are2p and Are1p fused to green fluorescent protein (GFP). Lipid analysis of nrrl and are2 deletion strains revealed that Are2p and A re1p utilize sterol substrates in vivo with different efficiency; Are2p has a significant preference for ergosterol as a substrate, whereas Are1p este rifies sterol precursors, mainly lanosterol, as well as ergosterol. The spe cificity towards fatty acids is similar for both isoenzymes. The lack of st eryl esters in are1are2 mutant cells is largely compensated by an increased level of free sterols. Nevertheless, terbinafine, an inhibitor of ergoster ol biosynthesis, inhibits growth of are1are2 cells more efficiently than gr owth of wild-type. In a growth competition experiment are1are2 cells grow m ore slowly than wild-type after several rounds of cultivation, suggesting t hat Are1p and Are2p or steryl esters, the product formed by these two enzym es, are mon important in the natural environment than under laboratory cond itions.