Functionality of the Seventh and Eighth Transmembrane Domains of Acyl-Coenzyme A:Cholesterol Acyltransferase 1
详细信息 查看全文
- 作者：Zhan-Yun Guo ; Catherine C. Y. Chang ; Ta-Yuan Chang
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：September 4, 2007
- 年：2007
- 卷：46
- 期：35
- 页码：10063 - 10071
- 全文大小：799K
- 年卷期：v.46,no.35(September 4, 2007)
- ISSN：1520-4995
文摘
Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) is a resident enzyme in theendoplasmic reticulum. ACAT1 is a homotetrameric protein and contains nine transmembrane domains(TMDs). His460 is a key active residue and is located within TMD7. Human ACAT1 has seven free Cys,but the recombinant ACAT1 devoid of free Cys retains full enzyme activity. To further probe thefunctionality of TMD7 (amino acids 446-460) and TMD8 (amino acids 466-481), we used a parentalACAT1 devoid of free Cys as the template to perform Cys-scanning mutagenesis within these regions.Each of the single Cys mutants was expressed in Chinese hamster ovary (CHO) cell line AC29 lackingendogenous ACAT1. We measured the effect of single Cys substitution on enzyme activity and used theCu(1,10-phenanthroline)2SO4-mediated disulfide cross-linking method to probe possible interactions ofengineered Cys between the two identical subunits. The results show that several residues in one subunitclosely interact with the same residues in the other subunit; mutating these residues to Cys does not leadto large loss in enzyme activity. Helical wheel analysis suggests that these residues are located at oneside of the coil. In contrast, mutating residues F453, A457, or H460 to Cys causes large loss in enzymeactivity; the latter residues are located at the opposite side of the coil. A similar arrangement is found forresidues in TMD8. Thus, helical coils in TMD7 and TMD8 have two distinct functional sides: one sideis involved in substrate-binding/catalysis, while the other side is involved in subunit interaction.