Human acyl-CoA:cholesterol acyltransferase-1 is a homotetrameric enzyme in intact cells and in vitro

Abstract

Acyl-CoA:cholesterol acyltransferase (ACAT) is a key enzyme in cellular cholesterol homeostasis and in atherosclerosis. ACAT-1 may function as an allosteric enzyme. We took a multifaceted approach to investigate the subunit composition of ACAT-1. When ACAT-1 with two different tags were co-expressed in the same Chinese hamster ovary cells, antibody specific to one tag caused co-immunoprecipitation of both types of ACAT-1 proteins. Radioimmunoprecipitations of cells expressing the untagged ACAT-1 or the 6- histidine-tagged ACAT-1 yielded a single radiolabeled band of predicted size on SDS-polyacrylamide gel electrophoresis. These results show that ACAT-1 exists as homo-oligomers in intact Chinese hamster ovary cells. We solubilized HisACAT-1 with the detergent deoxycholate or CHAPS (3-[(3- cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid), performed gel filtration chromatography and sucrose density gradient centrifugations in H2O and D2O, and determined the Stokes radii and sedimentation coefficients of the HisACAT1-detergent complexes. The estimated molecular mass of HisACAT- 1 is 263 kDa, which is 4 times that of the HisACAT-1 monomer (69 kDa). Finally, cross-linking experiments in intact cells and in vitro show that the increase in cross-linker concentrations causes an increase in size of the HisACAT1-positive signals, forming material(s) 4 times the size of the monomer, supporting the conclusion that ACAT-1 is a homotetrameric enzyme.