Department of Biochemistry and Cell Biology, Geisel School of Medicine, Dartmouth College, Hanover, NH, 03755, USA.
Department of Biochemistry and Cell Biology, Geisel School of Medicine, Dartmouth College, Hanover, NH, 03755, USA.
Arch Biochem Biophys. 2019 Aug 15;671:103-110. doi: 10.1016/j.abb.2019.06.006. Epub 2019 Jun 26.
Cholesterol is an important lipid molecule and is needed for all mammalian cells. In various cell types, excess cholesterol is stored as cholesteryl esters; acyl-CoA:cholesterol acyltransferase 1 (ACAT1) plays an essential role in this storage process. ACAT1 is located at the endoplasmic reticulum and has nine transmembrane domains (TMDs). It is a member of the membrane-bound O-acyltransferase (MBOAT) family, in which members contain multiple TMDs and participate in a variety of biological functions. When solubilized in the zwitterionic detergent CHAPS, ACAT1 can be purified to homogeneity with full enzyme activity and behaves as a homotetrameric protein. ACAT1 contains two dimerization motifs. The first motif is located near the N-terminus and is not conserved in MBOATs. Deletion of the N-terminal dimerization domain converts ACAT1 to a dimer with full catalytic activity; therefore, ACAT1 is a two-fold dimer. The second dimerization domain, located near the C-terminus, is conserved in MBOATs; however, it was not known whether the C-terminal dimerization domain is required for enzyme activity. Here we show that treating ACAT1 with non-ionic detergent, Triton X-100 or octyl glucoside, causes the enzyme to become a two-fold monomer without any enzymatic activity. Detergent exchange of Triton X-100 with CHAPS restores ACAT1 to a two-fold dimer but fails to restore its enzymatic activity. These results implicate that ACAT1 requires hydrophobic subunit interactions near the C-terminus in order to remain active as a two-fold dimer. Our results also caution the use of Triton X-100 or octyl glucoside to purify other MBOATs.
胆固醇是一种重要的脂质分子，所有哺乳动物细胞都需要它。在各种细胞类型中，过量的胆固醇被储存为胆固醇酯；酰基辅酶 A：胆固醇酰基转移酶 1（ACAT1）在这个储存过程中起着至关重要的作用。ACAT1 位于内质网，具有九个跨膜结构域（TMD）。它是膜结合酰基转移酶（MBOAT）家族的成员，该家族成员含有多个 TMD，并参与多种生物学功能。当在两性离子去污剂 CHAPS 中溶解时，ACAT1 可以被纯化至具有完全酶活性的均一状态，并表现为四聚体蛋白。ACAT1 包含两个二聚化基序。第一个基序位于 N 端附近，在 MBOAT 中不保守。删除 N 端二聚化结构域可将 ACAT1 转化为具有完全催化活性的二聚体；因此，ACAT1 是一个二倍体二聚体。第二个二聚化结构域位于 C 端附近，在 MBOAT 中保守；然而，尚不清楚 C 端二聚化结构域是否是酶活性所必需的。在这里，我们表明用非离子去污剂 Triton X-100 或辛基葡萄糖苷处理 ACAT1 会导致酶变成无任何酶活性的二倍体单体。用 CHAPS 交换 Triton X-100 可使 ACAT1 恢复为二倍体二聚体，但不能恢复其酶活性。这些结果表明，ACAT1 需要靠近 C 端的疏水性亚基相互作用才能保持其作为二倍体二聚体的活性。我们的结果还警告使用 Triton X-100 或辛基葡萄糖苷来纯化其他 MBOAT。
