Volume 272, Number 7, Issue of February 14, 1997 pp. 3980-3985
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Functional Expression of a cDNA to Human Acyl-coenzyme A:Cholesterol Acyltransferase in Yeast
SPECIES-DEPENDENT SUBSTRATE SPECIFICITY AND INHIBITOR SENSITIVITY

(Received for publication, September 23, 1996, and in revised form, November 11, 1996)

Hongyuan Yang , Debra Cromley ^§ , Hongxing Wang ^§ , Jeffrey T. Billheimer ^§ and Stephen L. Sturley

From the  Institute of Human Nutrition,  Departments of Pediatrics and Physiology and Molecular Biophysics, Columbia University College of Physicians and Surgeons, New York, New York 10032 and ^§ DuPont-Merck Research Laboratories, Experimental Station, Wilmington, Delaware 19880-0400

We have identified two yeast genes with similarity to a human cDNA encoding 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 yeast double mutant, resulting in high level production of ACAT protein, but low in vivo esterification of ergosterol, the predominant yeast sterol. The activity of the human enzyme was increased by incubation of these cells with 25-hydroxy, cholesterol, an established positive regulator of mammalian sterol esterification. In contrast, the yeast enzymes were unaffected by this reagent. In vitro microsomal assays indicated no sterol esterification in extracts from the double mutant. However, significant activity was detected from strains expressing human ACAT when cholesterol was equilibrated with the microsomal membranes. The human enzyme in yeast utilized cholesterol as the preferred sterol and was sensitive to competitive (S58035) and non-competitive (DuP 128) ACAT inhibitors. The yeast esterifying enzymes exhibited a diminished 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 preference for specific acyl-CoAs, particularly unsaturated C₁₈ forms. These results confirm the yeast genes as functional homologs of the human gene and demonstrate that the enzymes confer substrate specificity to the esterification reaction in both organisms.

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