The active site His-460 of human acyl-coenzyme A:cholesterol acyltransferase 1 resides in a hitherto undisclosed transmembrane domain.
Human acyl-coenzyme A:cholesterol acyltransferase 1 (hACAT1) esterifies cholesterol at the endoplasmic reticulum (ER). We had previously reported that hACAT1 contains seven transmembrane domains (TMD) (Lin, S., Cheng, D., Liu, M. S., Chen, J., and Chang, T. Y. (1999) J. Biol. Chem. 274, 23276-23285) and nine cysteines. The Cys near the N-terminal is located at the cytoplasm; the two cysteines near the C-terminal form a disulfide bond and are located in the ER lumen. The other six free cysteines are located in buried region(s) of the enzyme (Guo, Z.-Y., Chang, C. C. Y., Lu, X., Chen, J., Li, B.-L., and Chang, T.-Y. (2005) Biochemistry 44, 6537-6548). In the current study, we show that the conserved His-460 is a key active site residue for hACAT1. We next performed Cys-scanning mutagenesis within the region of amino acids 354-493, expressed these mutants in Chinese hamster ovary cells lacking ACAT1, and prepared microsomes from transfected cells. The microsomes are either left intact or permeabilized with detergent. The accessibility of the engineered cysteines of microsomal hACAT1 to various maleimide derivatives, including mPEG(5000)-maleimide (large, hydrophilic, and membrane-impermeant), N-ethylmaleimide, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (small, hydrophilic, and ER membrane-permeant), and N-phenylmaleimide (small, hydrophobic, and ER membrane-permeant), were monitored by Western blot analysis. The results led us to construct a revised, nine-TMD model, with the active site His-460 located within a hitherto undisclosed transmembrane domain, between Arg-443 and Tyr-462.
Guo
Zhan-Yun
ZY
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.
Lin
Song
S
Heinen
Jennifer A
JA
Chang
Catherine C Y
CC
Chang
Ta-Yuan
TY
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002462:Cell Membrane / Q000201:enzymology*; D006224:Cricetinae; D020869:Gene Expression Profiling; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary; D002785:Sterol O-Acyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D002785:Sterol O-Acyltransferase