Identification of phospholipid scramblase 1 as a novel interacting molecule with beta -secretase (beta -site amyloid precursor protein (APP) cleaving enzyme (BACE)).
Identification of phospholipid scramblase 1 as a novel interacting molecule with beta -secretase (beta -site amyloid precursor protein (APP) cleaving enzyme (BACE)).
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发表时间：
2003
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通讯作者：
S. Kametaka;M. Shibata;K. Moroe;S. Kanamori;Y. Ohsawa;S. Waguri;P. Sims;K. Emoto;M. Umeda;Y. Uchiyama
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作者：
S. Kametaka;M. Shibata;K. Moroe;S. Kanamori;Y. Ohsawa;S. Waguri;P. Sims;K. Emoto;M. Umeda;Y. Uchiyama
β-位点淀粉样前体蛋白(APP)裂解酶(BACE)是一种完整的膜天冬氨酸蛋白酶，负责APP的β-位点加工，其胞浆区域由24个氨基酸残基组成，参与BACE的内体定位。通过酵母双杂交筛选，我们发现磷脂混杂酶1的胞质结构域(PLSCR1)是一种II型整合膜蛋白，与BACE的胞浆区域相互作用。在培养的细胞中，BACE和PLSCR1共定位于高尔基体区和内体隔室，而U18666A处理后，BACE和PLSCR1共同分布在晚期内体来源的多囊体中，表明这两种蛋白在细胞内具有共同的转运途径。免疫共沉淀分析表明，在人神经母细胞瘤SH-SY5Y细胞中，这两种蛋白在内源表达水平上形成了蛋白质复合体，BACE尾部的二亮氨酸残基在体内外与PLSCR1的物理相互作用中也是必不可少的。此外，在SH-SY5Y细胞中，BACE和PLSCR1均定位于低浮力脂微区。二亮氨酸缺陷的BACE突变体也被分成脂质微区，但比野生型BACE要不稳定得多。综上所述，我们目前的研究提示PLSCR1的功能参与了BACE在细胞内的分布和/或BACE重新聚集到洗涤剂不溶的脂筏中。
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.