The outer membrane translocase (TOM) is the import channel for nuclear-encoded mitochondrial proteins. The general import pore contains Tom40, Tom22, Tom5, Tom6 and Tom7. Precursor proteins are bound by the peripheral receptor proteins Tom20, Tom22 and Tom70 before being imported by the TOM complex. Here we investigated the association of the receptor Tom20 with the TOM complex. Tom20 was found in the TOM complex, but not in a smaller subcomplex. In addition, a subcomplex was found without Tom40 and Tom7 but with Tom20. Using single particle tracking of labeled Tom20 in overexpressing human cells, we show that Tom20 has, on average, higher lateral mobility in the membrane than Tom7/TOM. After ligation of Tom20 with the TOM complex by post-tranlational protein trans-splicing using the trackless, ultra-fast cleaved Gp41-1 integrin system, a significant decrease in the mean diffusion coefficient of Tom20 was observed in the resulting Tom20-Tom7 fusion protein. Exposure of Tom20 to high substrate loading also resulted in reduced mobility. Taken together, our data show that the receptor subunit Tom20 interacts dynamically with the TOM core complex. We suggest that the TOM complex containing Tom20 is the active import pore and that Tom20 is associated when substrate is available.

外膜转位酶 (TOM) 是核编码线粒体蛋白的输入通道。一般导入孔包含Tom40、Tom22、Tom5、Tom6和Tom7。前体蛋白在被 TOM 复合物导入之前，与外周受体蛋白 Tom20、Tom22 和 Tom70 结合。在这里，我们研究了受体 Tom20 与 TOM 复合物的关联。在 TOM 复合体中发现了 Tom20，但在较小的亚复合体中没有发现。此外，还发现了一个没有 Tom40 和 Tom7 但有 Tom20 的亚群。在过度表达的人类细胞中使用标记的 Tom20 的单粒子跟踪，我们表明 Tom20 在膜中的横向迁移率平均高于 Tom7/TOM。在使用无轨超快速切割 Gp41-1 整合素系统通过翻译后蛋白质反式剪接将 Tom20 与 TOM 复合物连接后，在得到的 Tom20-Tom7 融合蛋白中观察到 Tom20 的平均扩散系数显着降低。Tom20 暴露于高基板负载也导致迁移率降低。总之，我们的数据显示受体亚基 Tom20 与 TOM 核心复合物动态相互作用。我们建议含有 Tom20 的 TOM 复合物是活性导入孔，并且当底物可用时，Tom20 是相关的。