BMC-Physiological Chemistry, LMU Munich, 82152 Martinsried, Germany.
Department of Biochemistry and Molecular Biology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
J Mol Biol. 2020 May 1;432(10):3326-3337. doi: 10.1016/j.jmb.2020.03.031. Epub 2020 Apr 8.
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.
在线粒体的膜间隙（IMS）中，Tim23 的受体结构域在数百种不同蛋白质从细胞质穿过 TOM 和 TIM23 复合物分别进入外膜和内膜的易位过程中具有重要作用。这个固有无序的结构域甚至可以延伸到细胞质中，在体外实验中，它被证明与 TOM 和 TIM23 复合物的几个亚基相互作用。为了从分子水平上理解这个 IMS 中的组织中心，我们在体内对 Tim23 的 IMS 结构域进行了剖析。我们发现，Tim23 与 Tim50 的相互作用面比之前认为的要大，并揭示了 Tim23 在 IMS 中与 Pam17 的意外相互作用，其功能障碍会影响它们在基质中的相互作用。此外，两个靠近内膜的保守负电荷残基的突变阻止了 Tim23 的二聚化。相同的突变增加了 Tim23 在线粒体表面的暴露，而膜电位的耗散则降低了它的暴露。我们的结果揭示了 Tim23 在 IMS 中复杂的相互作用网络，其影响可以通过两个线粒体膜传递，从而确保蛋白质有效地进入线粒体。
