Ukaji Koutarou, Ariyoshi Naoko, Sakaguchi Masao, Hamasaki Naotaka, Mihara Katsuyoshi
Department of Molecular Biology, Department of Clinical Chemistry and Laboratory Medicine, Graduate School of Medical Science, Kyushu University, Maidashi 3-1-1, Higashiku, Fukuoka 812-8582, Japan.
Biochem Biophys Res Commun. 2002 Mar 22;292(1):153-60. doi: 10.1006/bbrc.2002.6632.
We investigated the membrane topogenesis of glucose-6-phosphatase (G6Pase), a multispanning membrane protein, on the endoplasmic reticulum. In COS-7 cells, the first transmembrane segment (TM1) with weak hydrophobicity is inserted into the membrane in the N-terminus-out/C-terminus-cytoplasm orientation. The following TM2 is inserted depending on TM3. TM3 has the same orientation as TM1. In contrast to data from living cells, the full-length molecule and N-terminal fusion constructs were not inserted into the membrane in a cell-free system. Addition of a signal recognition particle did not improve G6Pase insertion. When the 37-residue N-terminal segment was deleted, however, TM2 and TM3 were correctly inserted. We concluded that the three N-terminal TM segments are inserted into the membrane dependent on the two signal-anchor sequences of TM1 and TM3. TM1 is likely to be an unconventional signal sequence that barely functions in vitro. The 37-residue N-terminal segment inhibits the signal function of the following TM3 in cell-free systems.
我们研究了内质网上多跨膜蛋白葡萄糖-6-磷酸酶（G6Pase）的膜拓扑形成过程。在COS-7细胞中，具有弱疏水性的第一个跨膜片段（TM1）以N端向外/C端在细胞质中的方向插入膜中。随后的TM2的插入取决于TM3。TM3与TM1具有相同的方向。与活细胞的数据相反，全长分子和N端融合构建体在无细胞系统中未插入膜中。添加信号识别颗粒并不能改善G6Pase的插入。然而，当删除37个氨基酸的N端片段时，TM2和TM3能正确插入。我们得出结论，三个N端跨膜片段依赖于TM1和TM3的两个信号锚定序列插入膜中。TM1可能是一个非传统的信号序列，在体外几乎不起作用。37个氨基酸的N端片段在无细胞系统中抑制了后续TM3的信号功能。
