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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Elsevier
2025-03-01
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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. |
format | Article |
id | doaj-art-7378d2c3a7224b97b57eef5ba792ba41 |
institution | Kabale University |
issn | 2666-1667 |
language | English |
publishDate | 2025-03-01 |
publisher | Elsevier |
record_format | Article |
series | STAR Protocols |
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 |
work_keys_str_mv | AT johnkim protocolforassessingthecloggingofthemitochondrialtranslocaseoftheoutermembranebyprecursorproteinsinhumancells AT hillaweidberg protocolforassessingthecloggingofthemitochondrialtranslocaseoftheoutermembranebyprecursorproteinsinhumancells |