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Triton X-100 or octyl glucoside inactivates acyl-CoA:cholesterol acyltransferase 1 by dissociating it from a two-fold dimer to a two-fold monomer

Neumann, Bryan, Chang, Catherine C.Y., Chang, Ta-Yuan
Archives of biochemistry and biophysics 2019 v.671 pp. 103-110
catalytic activity, cholesterol, cholesterol acyltransferase, cholesteryl esters, detergents, dimerization, endoplasmic reticulum, enzyme activity, glucosides, hydrophobicity, mammals, octoxynol, solubilization, zwitterions
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.