A simple method to disrupt and restore subunit interaction of acyl-CoA:cholesterol acyltransferase 1

Abstract

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. Method name: Dissociation of ACAT1 subunits by non-ionic detergents, Keywords: ACAT1, SOAT1, Two-fold dimer, MBOAT, Quaternary structure, Liposomes, Transmembrane, Cholesterol, Enzyme activity, Detergent