Two translocating hydrophilic segments of a nascent chain span the ER membrane during multispanning protein topogenesis
Two translocating hydrophilic segments of a nascent chain span the ER membrane during multispanning protein topogenesis
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DOI：
10.1083/jcb.200707050
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发表时间：
2007-12-31
影响因子：
7.8
通讯作者：
Sakaguchi, Masao
中科院分区：
文献类型：
--
作者：
Kida, Yuichiro;Morimoto, Fumiko;Sakaguchi, Masao
在蛋白质整合到内质网中的过程中，I型信号锚定序列之前的N-末端结构域通过易位子易位。通过将链霉亲和素结合肽标签融合到N末端，我们创建了多跨膜蛋白的整合中间体。在无细胞系统中，N-末端结构域（N-结构域）易位被链霉亲和素阻止，并被生物素恢复。即使当N-结构域易位被逮捕，第二疏水段介导的下游亲水段的易位。在一种定义的中间体中，两个亲水性片段和两个疏水性片段在生产状态下形成跨膜排列。两个易位的亲水性片段都与易位子亚基Sec 61 α交联。我们的结论是，在一个单一的膜蛋白中的两个易位的亲水性片段可以跨越跨膜在多跨越拓扑发生侧翼的易位子。此外，即使在六个连续的疏水片段进入易位子，N-结构域易位可以诱导重新启动从一个被捕的状态。这些观察结果表明了易位子非常灵活的性质。
During protein integration into the endoplasmic reticulum, the N-terminal domain preceding the type I signal-anchor sequence is translocated through a translocon. By fusing a streptavidin-binding peptide tag to the N terminus, we created integration intermediates of multispanning membrane proteins. In a cell-free system, N-terminal domain (N-domain) translocation was arrested by streptavidin and resumed by biotin. Even when N-domain translocation was arrested, the second hydrophobic segment mediated translocation of the downstream hydrophilic segment. In one of the defined intermediates, two hydrophilic segments and two hydrophobic segments formed a transmembrane disposition in a productive state. Both of the translocating hydrophilic segments were crosslinked with a translocon subunit, Sec61 alpha. We conclude that two translocating hydrophilic segment in a single membrane protein can span the membrane during multispanning topogenesis flanking the translocon. Furthermore, even after six successive hydrophobic segments entered the translocon, N-domain translocation could be induced to restart from an arrested state. These observations indicate the remarkably flexible nature of the translocon.