Department of Chemistry, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan.
Mol Biol Cell. 2012 Oct;23(20):3936-47. doi: 10.1091/mbc.E12-03-0202. Epub 2012 Aug 29.
The TOM40 complex is a protein translocator in the mitochondrial outer membrane and consists of several different subunits. Among them, Tom40 is a central subunit that constitutes a protein-conducting channel by forming a β-barrel structure. To probe the nature of the assembly process of Tom40 in the outer membrane, we attached various mitochondrial presequences to Tom40 that possess sorting information for the intermembrane space (IMS), inner membrane, and matrix and would compete with the inherent Tom40 assembly process. We analyzed the mitochondrial import of those fusion proteins in vitro. Tom40 crossed the outer membrane and/or inner membrane even in the presence of various sorting signals. N-terminal anchorage of the attached presequence to the inner membrane did not prevent Tom40 from associating with the TOB/SAM complex, although it impaired its efficient release from the TOB complex in vitro but not in vivo. The IMS or matrix-targeting presequence attached to Tom40 was effective in substituting for the requirement for small Tim proteins in the IMS for the translocation of Tom40 across the outer membrane. These results provide insight into the mechanism responsible for the precise delivery of β-barrel proteins to the outer mitochondrial membrane.
TOM40 复合物是线粒体外膜中的一种蛋白质转运体，由几个不同的亚基组成。其中，Tom40 是一个核心亚基，通过形成 β 桶结构构成蛋白质导通道。为了探究 Tom40 在 外膜中组装过程的本质，我们将具有跨膜空间（IMS）、内膜和基质分拣信息的各种线粒体前导序列附着到 Tom40 上，这些序列会与内在的 Tom40 组装过程竞争。我们在体外分析了这些融合蛋白的线粒体导入情况。Tom40 即使在存在各种分拣信号的情况下也能穿过外膜和/或内膜。附着的前导序列在 N 端锚定在内膜上并不会阻止 Tom40 与 TOB/SAM 复合物结合，尽管它会在体外而不是体内阻碍其从 TOB 复合物中的有效释放。附着在 Tom40 上的 IMS 或基质靶向前导序列可以有效地替代 IMS 中小 Tim 蛋白在 Tom40 跨外膜转运中的需求。这些结果为精确递送至线粒体外膜的 β 桶蛋白的机制提供了深入了解。
