Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
Proc Natl Acad Sci U S A. 2009 Dec 8;106(49):20800-5. doi: 10.1073/pnas.0910550106. Epub 2009 Nov 20.
Many bacterial proteins, including most secretory proteins, are translocated across the plasma membrane by the interplay of the cytoplasmic SecA ATPase and a protein-conducting channel formed by the SecY complex. SecA catalyzes the sequential movement of polypeptide segments through the SecY channel. How SecA interacts with a broad range of polypeptide segments is unclear, but structural data raise the possibility that translocation substrates bind into a "clamp" of SecA. Here, we have used disulfide bridge cross-linking to test this hypothesis. To analyze polypeptide interactions of SecA during translocation, two cysteines were introduced into a translocation intermediate: one that cross-links to the SecY channel and the other one for cross-linking to a cysteine placed at various positions in SecA. Our results show that a translocating polypeptide is indeed captured inside SecA's clamp and moves in an extended conformation through the clamp into the SecY channel. These results define the polypeptide path during SecA-mediated protein translocation and suggest a mechanism by which ATP hydrolysis by SecA is used to move a polypeptide chain through the SecY channel.
许多细菌蛋白，包括大多数分泌蛋白，都是通过细胞质 SecA ATP 酶和由 SecY 复合物形成的蛋白质传导通道的相互作用跨膜转运的。SecA 催化多肽片段通过 SecY 通道的顺序运动。SecA 如何与广泛的多肽片段相互作用尚不清楚，但结构数据提出了转运底物结合到 SecA“夹”中的可能性。在这里，我们使用二硫键交联来检验这一假设。为了分析 SecA 在转运过程中的多肽相互作用，我们在一个转运中间体内引入了两个半胱氨酸：一个与 SecY 通道交联，另一个与 SecA 中各种位置的半胱氨酸交联。我们的结果表明，一个正在转运的多肽确实被捕获在 SecA 的夹子里，并以伸展构象通过夹子里移动到 SecY 通道。这些结果定义了 SecA 介导的蛋白转运过程中的多肽路径，并提出了一种机制，即通过 SecA 的 ATP 水解来将多肽链通过 SecY 通道移动。
