Acyl-CoA:cholestereol acyltransferase 1 (ACAT1) is a two-fold dimer (homotetramer) and has two distinct dimerization domains. One domain is in an alpha-helical rich region near the cytoplasmic N-terminus. The other is proposed to be near the C-terminus where multiple transmembrane domains promote hydrophobic interactions between two ACAT1 subunits. The truncation of the ACAT1 N-terminal dimerization domain, Δ1-65, creates a dimer which is fully enzymatically active. It is currently not known how the C-terminal dimerization domain contributes to ACAT1 enzymatic activity. Here we describe a simple method that dissociates ACAT1 dimers through the addition of the non-ionic detergents Triton X-100 or octyl glucoside which disrupt the C-terminal dimerization domain. We also document the protocols for a method to exchange Triton X-100 with CHAPS to restore C-terminal dimerization of the ACAT1 protein, and an optimized liposomal assay to assess ACAT enzymatic activity. •This method can be applied to dissociate ACAT1 subunits by using Triton X-100 or octyl glucoside.•ACAT1 dimerization can be restored by exchanging Triton X-100 with CHAPS.•The liposomal ACAT activity assay conditions have been optimized.
Keywords: ACAT1; Cholesterol; Detergent; Dissociation of ACAT1 subunits by non-ionic detergents; Enzyme activity; Liposomes; MBOAT; Quaternary structure; SOAT1; Transmembrane; Two-fold dimer.
© 2019 The Authors.