Nohturfft A, Brown M S, Goldstein J L
Department of Molecular Genetics, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235, USA.
Proc Natl Acad Sci U S A. 1998 Oct 27;95(22):12848-53. doi: 10.1073/pnas.95.22.12848.
SREBP cleavage activating protein (SCAP), a membrane-bound glycoprotein, regulates the proteolytic activation of sterol regulatory element binding proteins (SREBPs), which are membrane-bound transcription factors that control lipid synthesis in animal cells. SCAP-stimulated proteolysis releases active fragments of SREBPs from membranes of the endoplasmic reticulum and allows them to enter the nucleus where they activate transcription. Sterols such as 25-hydroxycholesterol inactivate SCAP, suppressing SREBP proteolysis and turning off cholesterol synthesis. We here report the isolation of Chinese hamster ovary cells with a point mutation in SCAP (Y298C) that renders the protein resistant to inhibition by 25-hydroxycholesterol. Like the previously described D443N mutation, the Y298C mutation occurs within the putative sterol-sensing domain, which is part of the polytopic membrane attachment region of SCAP. Cells that express SCAP(Y298C) continued to process SREBPs in the presence of 25-hydroxycholesterol and hence they resisted killing by this sterol. In wild-type Chinese hamster ovary cells the N-linked carbohydrate chains of SCAP were mostly in the endoglycosidase H-sensitive form when cells were grown in medium containing 25-hydroxycholesterol. In contrast, when cells were grown in sterol-depleted medium, these chains were converted to an endoglycosidase H-resistant form. 25-Hydroxycholesterol had virtually no effect in cells expressing SCAP(D443N) or SCAP(Y298C). The relation between this regulated carbohydrate processing to the SCAP-regulated proteolysis of SREBP remains to be explored.
固醇调节元件结合蛋白裂解激活蛋白（SCAP）是一种膜结合糖蛋白，它调节固醇调节元件结合蛋白（SREBPs）的蛋白水解激活过程。SREBPs是膜结合转录因子，可控制动物细胞中的脂质合成。SCAP刺激的蛋白水解作用可从内质网的膜上释放SREBPs的活性片段，并使其进入细胞核，在细胞核中它们可激活转录。诸如25-羟基胆固醇之类的固醇可使SCAP失活，抑制SREBP的蛋白水解作用并关闭胆固醇合成。我们在此报告了分离出的中国仓鼠卵巢细胞，其SCAP存在一个点突变（Y298C），该突变使该蛋白对25-羟基胆固醇的抑制作用具有抗性。与先前描述的D443N突变一样，Y298C突变发生在假定的固醇感应域内，该区域是SCAP多跨膜附着区域的一部分。表达SCAP（Y298C）的细胞在存在25-羟基胆固醇的情况下继续处理SREBPs，因此它们对这种固醇的杀伤具有抗性。在野生型中国仓鼠卵巢细胞中，当细胞在含有25-羟基胆固醇的培养基中生长时，SCAP的N-连接糖链大多处于内切糖苷酶H敏感形式。相反，当细胞在固醇缺乏的培养基中生长时，这些糖链会转变为内切糖苷酶H抗性形式。25-羟基胆固醇对表达SCAP（D443N）或SCAP（Y298C）的细胞几乎没有影响。这种受调控的糖基化加工与SCAP调控的SREBP蛋白水解之间的关系仍有待探索。
