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The Mouse Heart Mitochondria N Terminome Provides Insights into ClpXP-Mediated Proteolysis

The mammalian mitochondrial proteome consists of more than 1100 annotated proteins and their proteostasis is regulated by only a few ATP-dependent protease complexes. Technical advances in protein mass spectrometry allowed for detailed description of the mitoproteome from different species and tissu...

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Autores principales: Hofsetz, Eduard, Demir, Fatih, Szczepanowska, Karolina, Kukat, Alexandra, Kizhakkedathu, Jayachandran N., Trifunovic, Aleksandra, Huesgen, Pitter F.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Biochemistry and Molecular Biology 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8014998/
https://www.ncbi.nlm.nih.gov/pubmed/32467259
http://dx.doi.org/10.1074/mcp.RA120.002082
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author Hofsetz, Eduard
Demir, Fatih
Szczepanowska, Karolina
Kukat, Alexandra
Kizhakkedathu, Jayachandran N.
Trifunovic, Aleksandra
Huesgen, Pitter F.
author_facet Hofsetz, Eduard
Demir, Fatih
Szczepanowska, Karolina
Kukat, Alexandra
Kizhakkedathu, Jayachandran N.
Trifunovic, Aleksandra
Huesgen, Pitter F.
author_sort Hofsetz, Eduard
collection PubMed
description The mammalian mitochondrial proteome consists of more than 1100 annotated proteins and their proteostasis is regulated by only a few ATP-dependent protease complexes. Technical advances in protein mass spectrometry allowed for detailed description of the mitoproteome from different species and tissues and their changes under specific conditions. However, protease-substrate relations within mitochondria are still poorly understood. Here, we combined Terminal Amine Isotope Labeling of Substrates (TAILS) N termini profiling of heart mitochondria proteomes isolated from wild type and Clpp(−/−) mice with a classical substrate-trapping screen using FLAG-tagged proteolytically active and inactive CLPP variants to identify new ClpXP substrates in mammalian mitochondria. Using TAILS, we identified N termini of more than 200 mitochondrial proteins. Expected N termini confirmed sequence determinants for mitochondrial targeting signal (MTS) cleavage and subsequent N-terminal processing after import, but the majority were protease-generated neo-N termini mapping to positions within the proteins. Quantitative comparison revealed widespread changes in protein processing patterns, including both strong increases or decreases in the abundance of specific neo-N termini, as well as an overall increase in the abundance of protease-generated neo-N termini in CLPP-deficient mitochondria that indicated altered mitochondrial proteostasis. Based on the combination of altered processing patterns, protein accumulation and stabilization in CLPP-deficient mice and interaction with CLPP, we identified OAT, HSPA9 and POLDIP2 and as novel bona fide ClpXP substrates. Finally, we propose that ClpXP participates in the cooperative degradation of UQCRC1. Together, our data provide the first landscape of the heart mitochondria N terminome and give further insights into regulatory and assisted proteolysis mediated by ClpXP.
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spelling pubmed-80149982021-04-12 The Mouse Heart Mitochondria N Terminome Provides Insights into ClpXP-Mediated Proteolysis Hofsetz, Eduard Demir, Fatih Szczepanowska, Karolina Kukat, Alexandra Kizhakkedathu, Jayachandran N. Trifunovic, Aleksandra Huesgen, Pitter F. Mol Cell Proteomics Research The mammalian mitochondrial proteome consists of more than 1100 annotated proteins and their proteostasis is regulated by only a few ATP-dependent protease complexes. Technical advances in protein mass spectrometry allowed for detailed description of the mitoproteome from different species and tissues and their changes under specific conditions. However, protease-substrate relations within mitochondria are still poorly understood. Here, we combined Terminal Amine Isotope Labeling of Substrates (TAILS) N termini profiling of heart mitochondria proteomes isolated from wild type and Clpp(−/−) mice with a classical substrate-trapping screen using FLAG-tagged proteolytically active and inactive CLPP variants to identify new ClpXP substrates in mammalian mitochondria. Using TAILS, we identified N termini of more than 200 mitochondrial proteins. Expected N termini confirmed sequence determinants for mitochondrial targeting signal (MTS) cleavage and subsequent N-terminal processing after import, but the majority were protease-generated neo-N termini mapping to positions within the proteins. Quantitative comparison revealed widespread changes in protein processing patterns, including both strong increases or decreases in the abundance of specific neo-N termini, as well as an overall increase in the abundance of protease-generated neo-N termini in CLPP-deficient mitochondria that indicated altered mitochondrial proteostasis. Based on the combination of altered processing patterns, protein accumulation and stabilization in CLPP-deficient mice and interaction with CLPP, we identified OAT, HSPA9 and POLDIP2 and as novel bona fide ClpXP substrates. Finally, we propose that ClpXP participates in the cooperative degradation of UQCRC1. Together, our data provide the first landscape of the heart mitochondria N terminome and give further insights into regulatory and assisted proteolysis mediated by ClpXP. American Society for Biochemistry and Molecular Biology 2020-11-23 /pmc/articles/PMC8014998/ /pubmed/32467259 http://dx.doi.org/10.1074/mcp.RA120.002082 Text en © 2020 © 2020 Hofsetz et al. http://creativecommons.org/licenses/by/4.0/ This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Research
Hofsetz, Eduard
Demir, Fatih
Szczepanowska, Karolina
Kukat, Alexandra
Kizhakkedathu, Jayachandran N.
Trifunovic, Aleksandra
Huesgen, Pitter F.
The Mouse Heart Mitochondria N Terminome Provides Insights into ClpXP-Mediated Proteolysis
title The Mouse Heart Mitochondria N Terminome Provides Insights into ClpXP-Mediated Proteolysis
title_full The Mouse Heart Mitochondria N Terminome Provides Insights into ClpXP-Mediated Proteolysis
title_fullStr The Mouse Heart Mitochondria N Terminome Provides Insights into ClpXP-Mediated Proteolysis
title_full_unstemmed The Mouse Heart Mitochondria N Terminome Provides Insights into ClpXP-Mediated Proteolysis
title_short The Mouse Heart Mitochondria N Terminome Provides Insights into ClpXP-Mediated Proteolysis
title_sort mouse heart mitochondria n terminome provides insights into clpxp-mediated proteolysis
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8014998/
https://www.ncbi.nlm.nih.gov/pubmed/32467259
http://dx.doi.org/10.1074/mcp.RA120.002082
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