Protocol for assessing the clogging of the mitochondrial translocase of the outer membrane by precursor proteins in human cells

• Published in: STAR protocols Vol. 6; no. 1; p. 103617
• Main Authors: Kim, John, Weidberg, Hilla
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 21.03.2025; Elsevier
• Subjects: cell biology; cell culture; Cell Fractionation - methods; cell separation/fractionation; Humans; Immunoprecipitation - methods; Mitochondria - metabolism; Mitochondrial Membranes - metabolism; Mitochondrial Precursor Protein Import Complex Proteins; molecular biology; protein biochemistry; protein expression and purification; Protein Precursors - metabolism; Protein Transport; Protocol; molecular biology; cell biology; protein biochemistry; cell separation/fractionation; cell culture; protein expression and purification
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2025.103617

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.1 [Display omitted] •Protocol to assess the stalling of mitochondrial precursors in TOM in human cell lines•Mitochondrial enrichment and protease protection assay determine precursor localization•Co-immunoprecipitation demonstrates precursor arrest inside TOM Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.