In the intermembrane space (IMS) of mitochondria, the receptor domain of Tim23 has an essential role during translocation of hundreds of different proteins from the cytosol via the TOM and TIM23 complexes in the outer and inner membranes, respectively. This intrinsically disordered domain, which can even extend into the cytosol, was shown, mostly in vitro, to interact with several subunits of the TOM and TIM23 complexes. To obtain molecular understanding of this organizational hub in the IMS, we dissected the IMS domain of Tim23 in vivo. We show that the interaction surface of Tim23 with Tim50 is larger than previously thought and reveal an unexpected interaction of Tim23 with Pam17 in the IMS, impairment of which influences their interaction in the matrix. Furthermore, mutations of two conserved negatively charged residues of Tim23, close to the inner membrane, prevented dimerization of Tim23. The same mutations increased exposure of Tim23 on the mitochondrial surface, whereas dissipation of membrane potential decreased it. Our results reveal an intricate network of Tim23 interactions in the IMS, whose influence is transduced across two mitochondrial membranes, ensuring efficient translocation of proteins into mitochondria.

中文翻译：

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Tim23的固有紊乱受体域的InVivo解剖。

在线粒体的膜间空间（IMS）中，Tim23的受体域在分别通过外膜和内膜中的TOM和TIM23复合物从细胞质中转移数百种不同蛋白质的过程中起着至关重要的作用。该内在无序的结构域，甚至可以延伸到胞质溶胶中，大部分在体外显示与TOM和TIM23复合体的几个亚基相互作用。为了获得对IMS中这个组织中心的分子理解，我们在体内解剖了Tim23的IMS域。我们显示，Tim23与Tim50的相互作用表面比以前认为的要大，并揭示了IMS中Tim23与Pam17的意外相互作用，其损伤影响了它们在基质中的相互作用。此外，Tim23的两个保守的带负电残基的突变 靠近内膜，防止Tim23二聚化。相同的突变增加了Tim23在线粒体表面的暴露，而膜电位的耗散降低了它。我们的研究结果揭示了IMS中Tim23相互作用的复杂网络，其影响跨两个线粒体膜传导，确保了蛋白质有效转移到线粒体中。