Functionality of the seventh and eighth transmembrane domains of acyl-coenzyme A:cholesterol acyltransferase 1.

摘要: Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) is a resident enzyme in the endoplasmic reticulum. ACAT1 homotetrameric protein and contains nine transmembrane domains (TMDs). His460 key active residue located within TMD7. Human has seven free Cys, but recombinant devoid of Cys retains full activity. To further probe functionality TMD7 (amino acids 446−460) TMD8 466−481), we used parental as template to perform Cys-scanning mutagenesis these regions. Each single mutants was expressed Chinese hamster ovary (CHO) cell line AC29 lacking endogenous ACAT1. We measured effect substitution on activity Cu(1,10-phenanthroline)2SO4-mediated disulfide cross-linking method possible interactions engineered between two identical subunits. The results show that several residues one subunit closely interact with same the...