Sato K, Mori H, Yoshida M, Tagaya M, Mizushima S
Research Laboratory of Resources Utilization, Tokyo Institute of Technology, Nagatsuta 4259, Yokohama 226, Japan.
J Biol Chem. 1997 Feb 28;272(9):5880-6. doi: 10.1074/jbc.272.9.5880.
Based on the finding that a series of engineered proOmpAs containing disulfide-bridged loops of different sizes at different positions exhibits a discontinuous mode of polypeptide transit across the cytoplasmic membrane of Escherichia coli, we suggested previously that the translocation of preproteins takes place at every 30 amino acid residues (Uchida, K., Mori, H., and Mizushima, S. (1995) J. Biol. Chem. 270, 30862-30868). In the present study, we investigated the molecular mechanism underlying this stepwise translocation. Deletion or relocation of hydrophobic segments of the mature domain of proOmpA (H1, residues 233-237; H2, residues 261-265) significantly altered the pattern of the stepwise translocation. The stepwise mode of polypeptide insertion was also observed with reconstituted proteoliposomes comprising purified SecA, SecY, and SecE. Cross-linking experiments involving a photoactivable cross-linker revealed that SecY and SecA are the components which interact with the hydrophobic segment of proOmpA. The present results indicate that the hydrophobic segments of the mature domains of preproteins interact with membrane embedded translocase during polypeptide transit across the membrane, which causes a discontinuous mode of polypeptide movement.
基于一系列在不同位置含有不同大小二硫键桥环的工程化前OmpA在大肠杆菌细胞质膜上呈现多肽跨膜转运的不连续模式这一发现，我们之前提出前体蛋白的转运每30个氨基酸残基发生一次（内田 健、森 浩、水岛 修（1995年）《生物化学杂志》270卷，30862 - 30868页）。在本研究中，我们探究了这种逐步转运背后的分子机制。前OmpA成熟结构域疏水片段（H1，第233 - 237位残基；H2，第261 - 265位残基）的缺失或重定位显著改变了逐步转运的模式。在由纯化的SecA、SecY和SecE组成的重组蛋白脂质体中也观察到了多肽插入的逐步模式。涉及光活化交联剂的交联实验表明，SecY和SecA是与前OmpA疏水片段相互作用的组分。目前的结果表明，前体蛋白成熟结构域的疏水片段在多肽跨膜转运过程中与膜嵌入的转位酶相互作用，这导致了多肽运动的不连续模式。
