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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.