Kuroiwa T, Sakaguchi M, Omura T, Mihara K
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan.
J Biol Chem. 1996 Mar 15;271(11):6423-8. doi: 10.1074/jbc.271.11.6423.
The reinitiation of the translocation of the growing nascent chain across the endoplasmic reticulum membrane is essential for the topogenesis of multispanning membrane proteins. We investigated the requirements for the reinitiation process using model proteins in which systematically designed sequences were inserted after two preceding topogenic sequences, namely the N-terminal signal sequence (S) and stop transfer sequence (St). The model proteins were translated in vitro in the presence of rough microsomes, and the final topology of the proteins in the microsomal membrane was examined by proteolytic digestion. The structural requirements for S and the reinitiation sequence (R) overlapped to some extent, but substantial differences were noticed. When St and R were separated by a short cytoplasmic segment (58 amino acids), the efficiency of the reinitiation was not affected by the concentration of the signal recognition particle (SRP) in the translation system, even though the sequence inserted as R was an SRP-dependent signal sequence. However, when the cytoplasmic segment was longer (100 amino acids), the reinitiation efficiency was reduced, and the SRP improved the overall efficiency as well as impaired the accessibility of the processing site after the R to the signal peptidase.
新生肽链跨内质网膜转运的重新起始对于多跨膜蛋白的拓扑结构形成至关重要。我们使用模型蛋白研究了重新起始过程的要求，这些模型蛋白在前面两个拓扑形成序列（即N端信号序列（S）和终止转移序列（St））之后插入了系统设计的序列。模型蛋白在糙面微粒体存在的情况下进行体外翻译，通过蛋白酶消化检查微粒体膜中蛋白质的最终拓扑结构。S和重新起始序列（R）的结构要求在一定程度上重叠，但也注意到了显著差异。当St和R被一个短的胞质片段（58个氨基酸）隔开时，重新起始效率不受翻译系统中信号识别颗粒（SRP）浓度的影响，尽管作为R插入的序列是一个依赖SRP的信号序列。然而，当胞质片段较长（100个氨基酸）时，重新起始效率降低，SRP提高了整体效率，但也损害了R之后加工位点对信号肽酶的可及性。
