Sakai J, Rawson R B, Espenshade P J, Cheng D, Seegmiller A C, Goldstein J L, Brown M S
Department of Molecular Genetics, University of Texas, Southwestern Medical Center, Dallas 75235, USA.
Mol Cell. 1998 Oct;2(4):505-14. doi: 10.1016/s1097-2765(00)80150-1.
The lipid composition of animal cells is controlled by SREBPs, transcription factors released from membranes by sterol-regulated proteolysis. Release is initiated by Site-1 protease (S1P), which cleaves SREBPs in the ER luminal loop between two membrane-spanning regions. To clone S1P, we prepared pCMV-PLAP-BP2, which encodes a fusion protein that contains placental alkaline phosphatase (PLAP) in the ER lumen flanked by cleavage sites for signal peptidase and S1P. In sterol-deprived cells, cleavage by both proteases leads to PLAP secretion. PLAP is not secreted by SRD-12B cells, cholesterol auxotrophs that lack S1P. We transfected SRD-12B cells with pCMV-PLAP-BP2 plus pools of CHO cDNAs and identified a cDNA that restores Site-1 cleavage and PLAP secretion. The cDNA encodes S1P, an intraluminal 1052-amino-acid membrane-bound subtilisin-like protease. We propose that S1P is the sterol-regulated protease that controls lipid metabolism in animal cells.
动物细胞的脂质组成受固醇调节元件结合蛋白（SREBPs）控制，SREBPs是一类通过固醇调节的蛋白水解作用从膜上释放的转录因子。释放过程由1号位点蛋白酶（S1P）启动，它在两个跨膜区域之间的内质网腔环中切割SREBPs。为了克隆S1P，我们制备了pCMV-PLAP-BP2，它编码一种融合蛋白，该蛋白在内质网腔中含有胎盘碱性磷酸酶（PLAP），两侧为信号肽酶和S1P的切割位点。在固醇缺乏的细胞中，两种蛋白酶的切割都会导致PLAP分泌。PLAP不会被SRD-12B细胞分泌，SRD-12B细胞是缺乏S1P的胆固醇营养缺陷型细胞。我们用pCMV-PLAP-BP2和中国仓鼠卵巢细胞（CHO）cDNA文库转染SRD-12B细胞，并鉴定出一个能恢复1号位点切割和PLAP分泌的cDNA。该cDNA编码S1P，一种腔内1052个氨基酸的膜结合枯草杆菌蛋白酶样蛋白酶。我们认为S1P是控制动物细胞脂质代谢的固醇调节蛋白酶。
