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1.
Plant Cell ; 31(1): 231-249, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30464037

RESUMO

The compartmentalization of PAPS (the sulfate donor 3'-phosphoadenosine 5'-phosphosulfate) synthesis (mainly in plastids), PAPS consumption (in the cytosol), and PAP (the stress signaling molecule 3'-phosphoadenosine 5'-phosphate) degradation (in plastids and mitochondria) requires organellar transport systems for both PAPS and PAP. The plastidial transporter PAPST1 (PAPS TRANSPORTER1) delivers newly synthesized PAPS from the stroma to the cytosol. We investigated the activity of PAPST2, the closest homolog of PAPST1, which unlike PAPST1 is targeted to both the plastids and mitochondria. Biochemical characterization in Arabidopsis thaliana revealed that PAPST2 mediates the antiport of PAP, PAPS, ATP, and ADP. Strongly increased cellular PAP levels negatively affect plant growth, as observed in the fry1 papst2 mutant, which lacks the PAP-catabolizing enzyme SALT TOLERANCE 1 and PAPST2. PAP levels were specifically elevated in the cytosol of papst2 and fiery1 papst2, but not in papst1 or fry1 papst1 PAPST1 failed to complement the papst2 mutant phenotype in mitochondria, because it likely removes PAPS from the cell, as demonstrated by the increased expression of phytosulfokine genes. Overexpression of SAL1 in mitochondria rescued the phenotype of fry1 but not fry1 papst2 Therefore, PAPST2 represents an important organellar importer of PAP, providing a piece of the puzzle in our understanding of the organelle-to-nucleus PAP retrograde signaling pathway.


Assuntos
Difosfato de Adenosina/metabolismo , Citosol/metabolismo , Plastídeos/metabolismo , Arabidopsis/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/metabolismo , Transdução de Sinais
2.
Mol Cell Proteomics ; 19(8): 1330-1345, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32467259

RESUMO

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.


Assuntos
Endopeptidase Clp/metabolismo , Mitocôndrias Cardíacas/metabolismo , Proteólise , Proteoma/metabolismo , Sequência de Aminoácidos , Animais , Endopeptidase Clp/deficiência , Camundongos , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Processamento de Proteína Pós-Traducional , Reprodutibilidade dos Testes , Especificidade por Substrato
3.
EMBO J ; 35(23): 2566-2583, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27797820

RESUMO

Despite being one of the most studied proteases in bacteria, very little is known about the role of ClpXP in mitochondria. We now present evidence that mammalian CLPP has an essential role in determining the rate of mitochondrial protein synthesis by regulating the level of mitoribosome assembly. Through a proteomic approach and the use of a catalytically inactive CLPP, we produced the first comprehensive list of possible mammalian ClpXP substrates involved in the regulation of mitochondrial translation, oxidative phosphorylation, and a number of metabolic pathways. We further show that the defect in mitoribosomal assembly is a consequence of the accumulation of ERAL1, a putative 12S rRNA chaperone, and novel ClpXP substrate. The presented data suggest that the timely removal of ERAL1 from the small ribosomal subunit is essential for the efficient maturation of the mitoribosome and a normal rate of mitochondrial translation.


Assuntos
Endopeptidase Clp/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Mitocôndrias/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Animais , Células Cultivadas , Fibroblastos/fisiologia , Camundongos , Camundongos Knockout , Biossíntese de Proteínas
4.
Methods Mol Biol ; 2192: 313-329, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33230781

RESUMO

Mitochondrial proteases constitute a fundamental part of the organellar protein quality control system to ensure the timely removal of damaged or obsolete proteins. The analysis of proteases is often limited to the identification of bona fide substrates that are degraded in the presence and become more abundant in the absence of the respective protease. However, proteases in numerous organisms from bacteria to humans can process specific substrates to release shortened proteins with potentially altered activities. Here, we describe an adaptation of the substrate-trapping approach, as well as the N-terminal profiling protocol Terminal Amine Isotope Labeling of Substrates (TAILS) for the identification of bona fide substrates and mitochondrial proteins that undergo complete or partial proteolysis.


Assuntos
Proteases Dependentes de ATP/química , Proteases Dependentes de ATP/metabolismo , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Proteases Dependentes de ATP/genética , Animais , Células Cultivadas , Cromatografia Líquida/métodos , Fibroblastos/citologia , Imunoprecipitação/métodos , Marcação por Isótopo/métodos , Camundongos , Camundongos Knockout , Miocárdio/citologia , Processamento de Proteína Pós-Traducional , Proteólise , Especificidade por Substrato , Espectrometria de Massas em Tandem/métodos , Transfecção/métodos
5.
Nat Commun ; 11(1): 1643, 2020 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-32242014

RESUMO

Regulation of the turnover of complex I (CI), the largest mitochondrial respiratory chain complex, remains enigmatic despite huge advancement in understanding its structure and the assembly. Here, we report that the NADH-oxidizing N-module of CI is turned over at a higher rate and largely independently of the rest of the complex by mitochondrial matrix protease ClpXP, which selectively removes and degrades damaged subunits. The observed mechanism seems to be a safeguard against the accumulation of dysfunctional CI arising from the inactivation of the N-module subunits due to attrition caused by its constant activity under physiological conditions. This CI salvage pathway maintains highly functional CI through a favorable mechanism that demands much lower energetic cost than de novo synthesis and reassembly of the entire CI. Our results also identify ClpXP activity as an unforeseen target for therapeutic interventions in the large group of mitochondrial diseases characterized by the CI instability.


Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Animais , Complexo I de Transporte de Elétrons/genética , Endopeptidase Clp/genética , Endopeptidase Clp/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mioblastos/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo
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