Guo Zhan-Yun, Lin Song, Heinen Jennifer A, Chang Catherine C Y, Chang Ta-Yuan
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.
J Biol Chem. 2005 Nov 11;280(45):37814-26. doi: 10.1074/jbc.M508384200. Epub 2005 Sep 8.
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.
人酰基辅酶A：胆固醇酰基转移酶1（hACAT1）在内质网（ER）中使胆固醇酯化。我们之前报道过hACAT1含有七个跨膜结构域（TMD）（林，S.，程，D.，刘，M.S.，陈，J.，和张，T.Y.（1999）《生物化学杂志》274，23276 - 23285）和九个半胱氨酸。靠近N端的半胱氨酸位于细胞质中；靠近C端的两个半胱氨酸形成二硫键，位于内质网腔中。其他六个游离半胱氨酸位于该酶的埋藏区域（郭，Z.-Y.，张，C.C.Y.，卢，X.，陈，J.，李，B.-L.，和张，T.-Y.（2005）《生物化学》44，6537 - 6548）。在当前研究中，我们表明保守的His - 460是hACAT1的关键活性位点残基。接下来，我们在氨基酸354 - 493区域内进行了半胱氨酸扫描诱变，在缺乏ACAT1的中国仓鼠卵巢细胞中表达这些突变体，并从转染细胞中制备微粒体。微粒体要么保持完整，要么用去污剂使其通透。通过蛋白质印迹分析监测微粒体hACAT1中工程化半胱氨酸对各种马来酰亚胺衍生物的可及性，这些衍生物包括mPEG(5000)-马来酰亚胺（大的、亲水性的且不能透过膜的）、N - 乙基马来酰亚胺、4 - 乙酰氨基 - 4'-马来酰亚胺基芪 - 2,2'-二磺酸（小的、亲水性的且能透过内质网膜的）以及N - 苯基马来酰亚胺（小的、疏水性的且能透过内质网膜的）。结果使我们构建了一个修订的九个跨膜结构域模型，活性位点His - 460位于一个迄今未公开的跨膜结构域内，在Arg - 443和Tyr - 462之间。
