Second-site cleavage in sterol regulatory element-binding protein occurs at transmembrane junction as determined by cysteine panning
Second-site cleavage in sterol regulatory element-binding protein occurs at transmembrane junction as determined by cysteine panning
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DOI：
10.1074/jbc.273.28.17801
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发表时间：
1998-07-10
影响因子：
4.8
通讯作者：
Brown, MS
中科院分区：
文献类型：
--
作者：
Duncan, EA;Davé, UP;Brown, MS
在响应甾醇剥夺时，两个连续的蛋白水解裂解从细胞膜释放甾醇调节元件结合蛋白（SREBP）的NH 2-末端片段，这些片段移位到细胞核，在那里它们激活参与胆固醇和脂肪酸代谢的基因。SREBP以发夹的方式结合到膜上。NH 2-末端和COOH-末端结构域面向细胞质，由两个跨膜节段和一个短的内腔环分开。第一次裂解发生在内腔环的位点1。然后通过在位点2处切割释放NH 2末端片段，该位点被认为位于第一跨膜区段内。在这里，我们使用一种新的半胱氨酸淘选方法，以确定在人类SREBP-2的第二个切割位点（站点-2）的Leu（484）Cys（485）键，位于之间的连接胞质NH 2-末端片段和第一个跨膜段。我们用编码融合蛋白的cDNA转染细胞，该融合蛋白在半胱氨酸485的NH 2-末端和COOH-末端侧的位置处具有单个半胱氨酸残基。测试NH 2-末端片段对用N-α-（3-马来酰亚胺基丙酰基）生物胞素修饰的敏感性，所述生物胞素将生物素基团连接到半胱氨酸巯基。半胱氨酸485的NH 2-末端侧的半胱氨酸保留在NH 2-末端片段上，但亮氨酸484的COOH-末端侧的半胱氨酸丢失。亮氨酸484是四肽Asp-Arg-Ser-Arg的COOH末端侧的三个残基，其紧接在第一跨膜区段之前并且是位点2切割所需的。
In response to sterol deprivation, two sequential proteolytic cleavages release the NH2-terminal fragments of sterol regulatory element-binding proteins (SREBPs) from cell membranes, The fragments translocate to the nucleus where they activate genes involved in cholesterol and fatty acid metabolism. The SREBPs are bound to membranes in a hairpin fashion. The NH2-terminal and COOH-terminal domains face the cytoplasm, separated by two membrane spanning segments and a short lumenal loop. The first cleavage occurs at Site-1 in the lumenal loop. The NH2-terminal fragment is then released by cleavage at Site-2, which is believed to lie within the first transmembrane segment. Here, we use a novel cysteine panning method to identify the second cleavage site (Site-2) in human SREBP-2 as the Leu(484) Cys(485) bond that lies at the junction between the cytoplasmic NH2-terminal fragment and the first transmembrane segment. We transfected cells with cDNAs encoding fusion proteins with single cysteine residues at positions to the NH2-terminal and COOH-terminal sides of cysteine 485. The NH2-terminal fragments were tested for susceptibility to modification with N-alpha-(3-maleimidylpropionyl)biocytin, which attaches a biotin group to cysteine sulfhydryls, Cysteines to the NH2-terminal side of cysteine 485 were retained on the NH2-terminal fragment, but cysteines to the COOH-terminal side of leucine 484 were lost. Leucine 484 is three residues to the COOH-terminal side of the tetrapeptide Asp-Arg-Ser-Arg, which immediately precedes the first transmembrane segment and is required for Site-2 cleavage.