Abteilung für Biochemie II, Universität Göttingen, D-37073 Göttingen, Germany.
J Cell Biol. 2011 Nov 14;195(4):643-56. doi: 10.1083/jcb.201105098. Epub 2011 Nov 7.
N-terminal targeting signals (presequences) direct proteins across the TOM complex in the outer mitochondrial membrane and the TIM23 complex in the inner mitochondrial membrane. Presequences provide directionality to the transport process and regulate the transport machineries during translocation. However, surprisingly little is known about how presequence receptors interact with the signals and what role these interactions play during preprotein transport. Here, we identify signal-binding sites of presequence receptors through photo-affinity labeling. Using engineered presequence probes, photo cross-linking sites on mitochondrial proteins were mapped mass spectrometrically, thereby defining a presequence-binding domain of Tim50, a core subunit of the TIM23 complex that is essential for mitochondrial protein import. Our results establish Tim50 as the primary presequence receptor at the inner membrane and show that targeting signals and Tim50 regulate the Tim23 channel in an antagonistic manner.
N 端靶向信号（前导序列）引导蛋白质穿过外膜中的 TOM 复合物和内膜中的 TIM23 复合物。前导序列为运输过程提供方向性，并在易位过程中调节运输机制。然而，令人惊讶的是，人们对前导序列受体如何与信号相互作用以及这些相互作用在前体蛋白运输过程中扮演什么角色知之甚少。在这里，我们通过光亲和标记来识别前导序列受体的信号结合位点。使用工程化的前导序列探针，通过质谱法对线粒体蛋白上的光交联位点进行了映射，从而定义了 TIM23 复合物核心亚基 Tim50 的前导序列结合域，Tim50 对于线粒体蛋白的输入是必不可少的。我们的结果将 Tim50 确立为内膜中的主要前导序列受体，并表明靶向信号和 Tim50 以拮抗的方式调节 Tim23 通道。
