Environmental Transition of Signal-Anchor Sequences during Membrane Insertion via the Endoplasmic Reticulum Translocon
Environmental Transition of Signal-Anchor Sequences during Membrane Insertion via the Endoplasmic Reticulum Translocon
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DOI：
10.1091/mbc.e09-08-0738
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发表时间：
2010-02-01
影响因子：
3.3
通讯作者：
Sakaguchi, Masao
中科院分区：
文献类型：
--
作者：
Kida, Yuichiro;Kume, Chisato;Sakaguchi, Masao
在内质网膜蛋白的生物发生过程中，一种被称为易位子的蛋白质传导通道在管腔结构域的膜易位和跨膜段的整合中都起着作用。在这里，我们分析了n -乙基马来酰亚胺在位置导向的Cys残基上的水依赖烷基化过程中多肽链的环境。利用该技术，可以检测到嵌入在膜疏水部分的信号锚定序列及其通过插入Pro残基而缩短的区域。当通过捕获n端融合的亲和标签序列阻止信号锚蛋白n端结构域的易位时，信号锚蛋白容易发生烷基化，这表明它向疏水环境的迁移也被阻止了。此外，当通过插入亲水性序列将标签序列与信号锚分离时，即使在n端捕获状态下，信号锚也无法进行烷基化。这表明信号锚蛋白的膜整合与其n端结构域的部分易位同步。此外，在膜蛋白的整合中间体中，这两条阻止易位的亲水链都位于易位子两侧的水环境中，这表明易位子提供了至少两条易位链的亲水性途径。
In biogenesis of membrane proteins on the endoplasmic reticulum, a protein-conducting channel called the translocon functions in both the membrane translocation of lumenal domains and the integration of transmembrane segments. Here we analyzed the environments of polypeptide chains during the processes by water-dependent alkylation of N-ethylmaleimide at site-directed Cys residues. Using the technique, the region embedded in the hydrophobic portion of the membrane within a signal-anchor sequence and its shortening by insertion of a Pro residue could be detected. When translocation of the N-terminal domain of the signal-anchor was arrested by trapping an N-terminally fused affinity tag sequence,the signal-anchor was susceptible to alkylation, indicating that its migration into the hydrophobic environment was also arrested. Furthermore, when the tag sequence was separated from the signal-anchor by insertion of a hydrophilic sequence,the signal-anchor became in accessible to alkylation even in the N-terminally trapped state. This suggests that membrane integration of the signal-anchor synchronizes with partial translocation of its N-terminal domain. Additionally, in an integration intermediate of a membrane protein, both of the two translocation-arrested hydrophilic chains were in an aqueous environment flanking the translocon, suggesting that the translocon provides the hydrophilic pathway capable of at least two translocating chains.