Identification of phospholipid scramblase 1 as a novel interacting molecule with β-secretase (β-site amyloid precursor protein (APP) cleaving enzyme (BACE))
Identification of phospholipid scramblase 1 as a novel interacting molecule with β-secretase (β-site amyloid precursor protein (APP) cleaving enzyme (BACE))
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DOI：
10.1074/jbc.m208611200
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发表时间：
2003-04-25
影响因子：
4.8
通讯作者：
Uchiyama, Y
中科院分区：
文献类型：
--
作者：
Kametaka, S;Shibata, M;Uchiyama, Y
β -site amyloid precursor protein (APP)- cleaved enzyme （BACE）是一种完整的膜天门氨酸蛋白酶，负责APP的β -site加工，其胞质区由24个氨基酸残基组成，参与了BACE的内体定位。通过酵母双杂交筛选，我们发现II型整合膜蛋白磷脂合成酶1 （PLSCR1）的胞质区域与BACE的胞质区域相互作用。在培养细胞中，BACE和PLSCR1共定位于高尔基体区和内体室，而当用U18666A处理时，它们在内体衍生的晚期多泡体中共重新分布，这表明这两种蛋白在细胞中共享一个共同的运输途径。共免疫沉淀分析显示，这两种蛋白在人神经母细胞瘤sh - sy5y细胞中形成内源性表达水平的蛋白复合物，BACE尾部的二苯甲酸残基也被发现是体外和体内与PLSCR1物理相互作用所必需的。此外，BACE和PLSCR1都定位于SH-SY5Y细胞的低浮力脂质微域。二亮氨酸缺陷型BACE突变体也被分离到脂质微结构域，但稳定性远不如野生型BACE。综上所述，我们目前的研究表明PLSCR1在BACE的细胞内分布和/或BACE募集到洗涤剂不溶性脂质筏中的功能参与。
beta-Site amyloid precursor protein (APP)-cleaving enzyme (BACE) is an integral membrane aspartic proteinase responsible for beta-site processing of APP, and its cytoplasmic region composed of 24 amino acid residues has been shown to be involved in the endosomal localization of BACE. With the yeast two-hybrid screening, we found that the cytoplasmic domain of phospholipid scramblase 1 (PLSCR1), a type II integral membrane protein, interacts with the cytoplasmic region of BACE. In cultured cells, BACE and PLSCR1 were colocalized in the Golgi area and in endosomal compartments, whereas they were co-redistributed in late endosome-derived multivesicular bodies when treated with U18666A, suggesting that both proteins share a common trafficking pathway in cells. Co-immunoprecipitation analysis showed that both proteins form a protein complex at an endogenous expression level in the human neuroblastoma SH-SY5Ycells, and the dileucine residue of the BACE tail is also revealed to be essential for the physical interaction with PLSCR1 in vitro and in vivo. Moreover, both BACE and PLSCR1 were localized in a low buoyant lipid microdomain in SH-SY5Y cells. The dileucine-defective BACE mutant was also fractionated into the lipid microdomain, but much less stably than wild-type BACE. Taken together, our current study suggests the functional involvement of PLSCR1 in the intracellular distribution of BACE and/or recruitment of BACE into the detergent-insoluble lipid raft.