Regulated cleavage of sterol regulatory element binding proteins requires sequences on both sides of the endoplasmic reticulum membrane
Regulated cleavage of sterol regulatory element binding proteins requires sequences on both sides of the endoplasmic reticulum membrane
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DOI：
10.1074/jbc.271.17.10379
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发表时间：
1996-04-26
影响因子：
4.8
通讯作者：
Goldstein, JL
中科院分区：
文献类型：
--
作者：
Hua, XX;Sakai, J;Goldstein, JL
固醇调节元件结合蛋白（SREBP-1和SREBP-2）通过发夹结构域附着于内质网（ER）和核膜，所述发夹结构域由通过类似于30个亲水性氨基酸的短内腔环连接的两个跨膜区组成。在固醇耗尽的细胞中，蛋白酶在第一跨膜结构域的区域中切割蛋白质，释放类似于500个氨基酸的NH 2末端片段，其激活编码低密度脂蛋白受体和胆固醇合成酶的基因的转录。在固醇超载的细胞中，蛋白质水解不发生，转录被抑制。通过对转染细胞的突变分析，我们确定了蛋白水解所需的两个SREBP片段，分别位于ER膜的两侧。腔内循环中的精氨酸是必不可少的。最大切割也需要邻近第一跨膜结构域的胞质表面上的四肽序列（DRSR）。这两个元件在人和仓鼠版本的SREBP-1和SREBP-2中是保守的。发现固醇介导的对SREBP-1切割的抑制依赖于末端COOH-末端区域（残基1034至COOH末端），其作为选择性剪接的结果以两种形式存在。由"a"类外显子（外显子18 a和19 a）编码的形式经历固醇调节的切割。由"c"类外显子（18 c和19 c）编码的形式切割效率较低，并且不受固醇抑制。这些研究是通过使用一种载体，实现低水平表达的表位标记的SREBP的控制下，相对较弱的胸苷激酶启动子从单纯疱疹病毒。与由高水平表达载体过量产生的SREBP相反，以低水平产生的SREBP受到与内源SREBP相同的调节切割模式。这些结果表明，甾醇调节的SREBP的蛋白水解是一个复杂的过程，需要在ER膜两侧的序列。
Sterol regulatory element binding proteins (SREBP-1 and SREBP-2) are attached to the endoplasmic reticulum (ER) and nuclear envelope by a hairpin domain consisting of two transmembrane regions connected by a short lumenal loop of similar to 30 hydrophilic amino acids. In sterol-depleted cells, a protease cleaves the protein in the region of the first transmembrane domain, releasing an NH2-terminal fragment of similar to 500 amino acids that activates transcription of genes encoding the low density lipoprotein receptor and enzymes of cholesterol synthesis. In sterol-overloaded cells, proteolysis does not occur, and transcription is repressed. Through mutational analysis in transfected cells, we identify two segments of SREBPs that are required for proteolysis, one on either side of the ER membrane. An arginine in the lumenal loop is essential. A tetrapeptide sequence (DRSR) on the cytosolic face adjacent to the first transmembrane domain is also required for maximal cleavage. Both of these elements are conserved in the human and hamster versions of SREBP-1 and SREBP-2. Sterol-mediated suppression of cleavage of SREBP-1 was found to be dependent on the extreme COOH-terminal region (residue 1034 to the COOH terminus), which exists in two forms as a result of alternative splicing. The form encoded by the ''a'' class exons (exons 18a and 19a) undergoes sterol-regulated cleavage. The form encoded by the ''c'' class exons (18c and 19c) is cleaved less efficiently and is not suppressed by sterols. These studies were made possible through use of a vector that achieves low level expression of epitope-tagged SREBPs under control of the relatively weak thymidine kinase promoter from herpes simplex virus. In contrast to SREBPs overproduced by high level expression vectors, the SREBPs produced at low levels were subject to the same regulated cleavage pattern as the endogenous SREBPs. These results indicate that sterol-regulated proteolysis of SREBPs is a complex process, requiring sequences on both sides of the ER membrane.