Acyl-coenzyme A:cholesterol acyltransferase (ACAT)2 is a membrane-bound enzyme present in a variety of tissues and cells. It is mainly located at the endoplasmic reticulum, and catalyzes the biosynthesis of cholesteryl esters, using long-chain fatty acyl-coenzyme A and cholesterol as its substrates. ACAT plays important roles in cholesterol homeostasis. At the single cell level, it is a key enzyme that prevents excess free cholesterol from building up in the cell membranes. At the physiological level, it contributes cholesteryl esters as part of the neutral lipid cargo, to be packaged into the cores of very low density lipoproteins and chylomicrons. Under pathophysiological condition, in cholesterol-loaded macrophages, ACAT converts excess cholesterol into cholesteryl esters. This action reduces the amount of cholesterol available from the macrophage cell surface for efflux and converts the macrophages to foam cells, which are the hallmark of early lesions of the disease atherosclerosis (reviewed in Ref. 1). For these reasons, ACAT has been a drug target for pharmaceutical intervention of diseases, including atherosclerosis and hyperlipidemia. In mammals, two ACAT genes exist that encode for two similar but different proteins, ACAT1 and ACAT2. Available evidence suggests that ACAT1 and ACAT2 may function in distinct and complementary manners in various tissues (reviewed in Refs. 2 and 3). Unlike many other enzymes/proteins involved in cellular cholesterol metabolism, neither ACAT1 nor ACAT2 is regulated at the transcription level by the cholesterol-dependent SREBP (sterol regulatory element-binding protein) cleavage-activating protein (SCAP)/sterol regulatory element-binding protein pathway. Instead, available evidence suggests that ACAT1 may contain a distinct regulatory site that specifically recognizes cholesterol as its activator (4, 5). This mechanism allows ACAT1 to be up-regulated rapidly (within minutes) by cholesterol that builds up at the ER. The enzymological and biochemical characteristics of ACAT2 significantly diverge from those of ACAT1 in several ways; however, ACAT2 may also be allosterically regulated by cholesterol (5, 6).Molecular cloning of the human ACAT1 (hACAT1) gene (7) provided the opportunity to study its biochemical properties. The recombinant hACAT1 expressed in Chinese hamster ovary cells can be purified to homogeneity (8). However, due to the low quantities of protein derived from the purification process, current efforts in our laboratory focus on studies at the enzymological and cell biological levels, but not at the structural biology level. ACAT1 is homotetrameric in vitro and in intact cells (9). The region near the N-terminal contains a dimerization motif. Deleting the N-terminal region converts the enzyme into a homodimer; the dimeric enzyme is fully active catalytically, and remains to be allosterically regulated by cholesterol (10). ACAT1 contains multiple transmembrane domains (TMDs). To deduce its membrane topology, we had previously inserted the nine-amino acid HA tag a...