Kim John, Weidberg Hilla
Life Sciences Institute, Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.
STAR Protoc. 2025 Mar 21;6(1):103617. doi: 10.1016/j.xpro.2025.103617. Epub 2025 Jan 31.
Protein import into the mitochondria is required for organellar function. Inefficient import can result in the stalling of mitochondrial precursors inside the translocase of the outer membrane (TOM) and blockage of the mitochondrial entry gate. Here, we present a protocol to assess the clogging of TOM by mitochondrial precursors in human cell lines. We describe how the localization of mitochondrial precursors can be determined by cellular fractionation. We then show how co-immunoprecipitation can be used to test the stalling of precursors inside TOM. For complete details on the use and execution of this protocol, please refer to Kim et al..
蛋白质导入线粒体是细胞器功能所必需的。导入效率低下会导致线粒体前体在外膜转位酶（TOM）内停滞，并阻塞线粒体的入口。在这里，我们提出了一种评估人类细胞系中线粒体前体对TOM阻塞情况的方案。我们描述了如何通过细胞分级分离来确定线粒体前体的定位。然后，我们展示了如何使用免疫共沉淀来检测前体在TOM内的停滞情况。有关该方案的使用和执行的完整详细信息，请参考Kim等人的研究。
