FUNCTIONAL EXPRESSION OF A CDNA TO HUMAN ACYL-COENZYME A-CHOLESTEROL ACYLTRANSFERASE IN YEAST - SPECIES-DEPENDENT SUBSTRATE-SPECIFICITY ANDINHIBITOR SENSITIVITY

We have identified two yeast genes with similarity to a human cDNA enc oding acyl-coenzyme A:cholesterol acyltransferase (ACAT), Deletion of both yeast genes results in a viable cell with undetectable esterified sterol (Yang, H., Bard, M., Bruner, D. A., Gleeson, A., Deckelbaum, R . J., Aljinovic, G., Pohl, T., Rothstein, R., and Sturley, S. L. (1996 ) Science 272, 1353-1356). Here, we expressed the human cDNA in the ye ast double mutant, resulting in high level production of ACAT protein, but low in vivo esterification of ergosterol, the predominant yeast s terol, The activity of the human enzyme was increased by incubation of these cells with 25-hydroxy, cholesterol, an established positive reg ulator of mammalian sterol esterification. In contrast, the yeast enzy mes were unaffected by this reagent. In vitro microsomal assays indica ted no sterol esterification in extracts from the double mutant. Howev er, significant activity was detected from strains expressing human AC AT when cholesterol was equilibrated with the microsomal membranes, Th e human enzyme in yeast uti lized cholesterol as the preferred sterol and was sensitive to competitive (S58035) and non-competitive (DuP 128 ) ACAT inhibitors, The yeast esterifying enzymes exhibited a diminishe d sterol substrate preference and were sensitive only to S58035, Human ACAT had a broad acyl-CoA substrate specificity, the other substrate for this reaction. By contrast, the yeast enzymes had a marked prefere nce for specific acyl-CoAs, particularly unsaturated C-18 forms. These results confirm the yeast genes as functional homologs of the human g ene and demonstrate that the enzymes confer substrate specificity to t he esterification reaction in both organisms.