期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 280, 期 45, 页码 37814-37826出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M508384200
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资金
- NHLBI NIH HHS [HL60306] Funding Source: Medline
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 His460 located within a hitherto undisclosed transmembrane domain, between Arg-443 and Tyr-462.
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