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

Summary: 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 clog...

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Main Authors: John Kim, Hilla Weidberg
Format: Article
Language:English
Published: Elsevier 2025-03-01
Series:STAR Protocols
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Online Access:http://www.sciencedirect.com/science/article/pii/S2666166725000231
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author John Kim
Hilla Weidberg
author_facet John Kim
Hilla Weidberg
author_sort John Kim
collection DOAJ
description Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
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spelling doaj-art-7378d2c3a7224b97b57eef5ba792ba412025-02-02T05:29:21ZengElsevierSTAR Protocols2666-16672025-03-0161103617Protocol for assessing the clogging of the mitochondrial translocase of the outer membrane by precursor proteins in human cellsJohn Kim0Hilla Weidberg1Life Sciences Institute, Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC V6T 1Z3, Canada; Corresponding authorLife Sciences Institute, Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC V6T 1Z3, Canada; Corresponding authorSummary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.http://www.sciencedirect.com/science/article/pii/S2666166725000231cell biologycell culturecell separation/fractionationmolecular biologyprotein biochemistryprotein expression and purification
spellingShingle John Kim
Hilla Weidberg
Protocol for assessing the clogging of the mitochondrial translocase of the outer membrane by precursor proteins in human cells
STAR Protocols
cell biology
cell culture
cell separation/fractionation
molecular biology
protein biochemistry
protein expression and purification
title Protocol for assessing the clogging of the mitochondrial translocase of the outer membrane by precursor proteins in human cells
title_full Protocol for assessing the clogging of the mitochondrial translocase of the outer membrane by precursor proteins in human cells
title_fullStr Protocol for assessing the clogging of the mitochondrial translocase of the outer membrane by precursor proteins in human cells
title_full_unstemmed Protocol for assessing the clogging of the mitochondrial translocase of the outer membrane by precursor proteins in human cells
title_short Protocol for assessing the clogging of the mitochondrial translocase of the outer membrane by precursor proteins in human cells
title_sort protocol for assessing the clogging of the mitochondrial translocase of the outer membrane by precursor proteins in human cells
topic cell biology
cell culture
cell separation/fractionation
molecular biology
protein biochemistry
protein expression and purification
url http://www.sciencedirect.com/science/article/pii/S2666166725000231
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