PHB1 and PHB2 deletion cell proteome
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Exploris 480
ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)
TISSUE(S): Cell Suspension Culture
SUBMITTER: 
Hendrik Nolte
LAB HEAD: Thomas Langer
PROVIDER: PXD043668 | Pride | 2023-08-14
REPOSITORIES: Pride
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Project description:Folding of newly synthesized proteins poses challenges for a functional proteome. Dedicated protein quality control (PQC) systems promote either the folding of nascent polypeptides at ribosomes or, if this fails, ensure their degradation. While well studied for cytosolic protein biogenesis, it is not understood how these processes work for mitochondrially-encoded proteins, key subunits of the oxidative phosphorylation system (OXPHOS). Here, we identify dedicated hubs in proximity to mitoribosomal tunnel exits coordinating mitochondrial translation, protein import, assembly, and protein turnover. Conserved prohibitin/m-AAA protease supercomplexes and the availability of assembly chaperones determine the fate of newly synthesized proteins by molecular triaging. The localization of these competing activities in vicinity to the mitoribosomal tunnel exit allows for a prompt decision whether newly synthesized proteins are fed into OXPHOS assembly or degraded. This repository contains the results of the FLAG-based enrichment of the native prohibitin complex. Please note that we measured elution either with LDS sample buffer or using FLAG-peptides. In the published study, the LDS elution buffer was shown.
2023-08-14 | PXD043671 | Pride
Project description:The mitochondrial oxidative phosphorylation system (OXPHOS) contains subunits of dual genetic origin, namely the nuclear and the mitochondrial genome. Mounting the different subunits into functional OXPHOS complexes is aided by dedicated chaperones termed assembly factors. How unfunctional or superfluous proteins are recognized during assembly and directed to degradation is currently not understood. Here, we reveal that protein quality control is an integral part of assembly, which is organized in dedicated hubs gathering mitochondrial translation, protein import, assembly, and protein turnover. Fate-decision of newly synthesized proteins is achieved by molecular triaging engaging the evolutionary conserved prohibitin/m-AAA protease supercomplex and assembly chaperones. The localization of these competing activities in vicinity to the mitoribosomal tunnel exit allows for a rapid decision whether newly synthesized proteins are fed into OXPHOS assembly or to degradation. In total, three projects are hosted in this repository. 1) Phb1-Alfa DSG cross-linked immunoprecipitation 2) Metabolic switch from Glycerol to Glucose followed by Phb1-ALFA DSG cross-linked immunoprecipitation. 3) Phb2-ALFA DSG cross-linked immunoprecipitation.
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Project description:Folding of newly synthesized proteins to the native state is a major challenge within the crowded cellular environment, since non-productive interactions can lead to misfolding, aggregation and degradation1. Cells cope with this challenge by coupling synthesis with polypeptide folding and by employing molecular chaperones to safeguard folding already cotranslationally2. However, little is known about the final step of folding, the assembly of polypeptides into complexes, although most of the cellular proteome forms oligomeric assemblies3. In prokaryotes, a proof-of-concept study showed that assembly of heterodimeric luciferase is an organized cotranslational process, facilitated by spatially confined translation of the subunits encoded on a polycistronic mRNA4. In eukaryotes, however, fundamental differences such as rarity of polycistronic mRNAs and different chaperone constellations raise the question whether assembly is also coordinated with translation. Here we provide a systematic and mechanistic analysis of protein complex assembly in eukaryotes using ribosome profiling. We determined the in vivo nascent subunits interactions of 12 hetero-oligomeric protein complexes of Saccharomyces cerevisiae at near-residue resolution. We find 9 complexes assemble cotranslationally; the 3 complexes that do not show cotranslational interactions are regulated by dedicated assembly chaperones5-7. Cotranslational assembly often occurs uni-directionally, with one fully synthesized subunit engaging its nascent partner subunit(s), thereby counteracting its aggregation propensity. The onset of cotranslational subunit association coincides sharply with full exposure of the nascent interaction domain at the ribosomal tunnel exit. The action of the ribosome-associated Hsp70 chaperone Ssb8 is coordinated with assembly. Ssb transiently engages partially synthesized interaction domains, then dissociates before the onset of partner subunit association, presumably to prevent premature assembly interactions. Our study shows that cotranslational subunit association is a prevalent mechanism for hetero-oligomers assembly in yeast and indicates that translation, folding and assembly of protein complexes are integrated processes in eukaryotes.
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Project description:Mitochondrial gene expression relies on mitoribosomes to translate mitochondrial mRNAs. The biogenesis of mitoribosomes is an intricate process involving multiple assembly factors. Among these factors, GTP-binding proteins (GTPBPs) play crucial roles. In bacterial systems, numerous GTPBPs are required for ribosome subunit maturation, with EngB being a GTPBP involved in the ribosomal large subunit assembly. In this study, we focused on exploring the function of GTPBP8, the human homolog of EngB. We found that ablation of GTPBP8 leads to the inhibition of mitochondrial translation, resulting in significant impairment of oxidative phosphorylation. In the absence of GTPBP8, numerous mitoribosomal proteins from both subunits become destabilized, indicating GTPBP8's involvement in synchronized subunit assembly. Furthermore, structural analysis of mitoribosomes from GTPBP8 knock-out cells showed the accumulation of mitoribosomal large subunit assembly intermediates that are incapable of forming functional monosomes. Our study highlights the crucial role of GTPBP8 as a component of the mitochondrial gene expression machinery involved in mitoribosome biogenesis.
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Project description:The metabolic plasticity of mitochondria supports cell development and differentiation and ensures cell survival under stress. The peptidase OMA1 regulates mitochondrial morphology and stress signaling and orchestrates tumorigenesis and cell fate decisions in a cell and tissue-specific manner. Here, we have used unbiased systemic approaches to demonstrate that OMA1-dependent cell fate decisions are under metabolic control. A metabolism-focus CRISPR screen combined with an integrated analysis of human expression data unraveled a protective role of OMA1 against DNA damage. Nucleotide deficiencies induced by chemotherapeutic agents promote the selective, p53-dependent apoptosis of cells lacking OMA1. The protective effect of OMA1 does not depend on OMA1 activation nor on OMA1-mediated OPA1 and DELE1 processing. OMA1-deficient cells have reduced glycolytic capacity and accumulate OXPHOS proteins upon DNA damage. OXPHOS inhibition restores glycolysis and confers resistance against DNA damage. Thus, metabolic cues determine cell fate decisions by OMA1, which sheds new light on the role of OMA1 in cancerogenesis.
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Project description:Biogenesis of eukaryotic messenger ribonucleoprotein complexes (mRNPs) involves the synthesis, splicing, and 3’-processing of pre-mRNA, and the assembly of mature mRNPs for nuclear export. We mapped 23 mRNP biogenesis factors onto the newly synthesized yeast transcriptome, providing ~10^5-10^6 high-confidence RNA interaction sites per factor.
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2022-07-27 | PXD031641 | Pride
Project description:Coenzyme Q (or ubiquinone) is a redox-active lipid, which serves as universal electron carrier in the mitochondrial respiratory chain and antioxidant in the plasma membrane limiting lipid peroxidation and ferroptosis. Mechanisms allowing cellular coenzyme Q distribution after synthesis within mitochondria are not understood. Here, we identify the cytosolic lipid transfer protein STARD7 as a critical and limiting factor of intracellular coenzyme Q transport. Dual localization of STARD7 to the intermembrane space of mitochondria and the cytosol upon cleavage by the rhomboid protease PARL ensures the synthesis of coenzyme Q in mitochondria and its transport to the plasma membrane. While cytosolic STARD7 overexpression protects against ferroptosis, it limits CoQ abundance in mitochondria and respiratory cell growth, illustrating the need to coordinate CoQ synthesis and cellular distribution by PARL-mediated STARD7 processing. Our findings highlight the antioxidant function of mitochondrial CoQ against ferroptosis and identify PARL and STARD7 as promising targets to interfere with ferroptosis. The repository contains raw files and methods for project 0112 (brain – whole proteomics) and 0176 (spatial proteomics). The labeling of the raw files and the experiment – raw file matches are available in the individual search zip files. The individual sections are accompanied with the project identifier.
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Project description:During the co-translational assembly of protein complexes, a fully synthesized subunit engages with the nascent chain of a newly synthesized interaction partner. Such events are thought to contribute to productive assembly, but their exact physiological relevance remains underexplored. Here, we examined structural motifs contained in nucleoporins for their potential to facilitate co-translational assembly. We experimentally tested candidate structural motifs and identified several previously unknown co-translational interactions. We demonstrate by selective ribosome profiling that domain invasion motifs of beta-propellers, coiled-coils, and short linear motifs act as co-translational assembly domains. Such motifs are often contained in proteins that are members of multiple complexes (moonlighters) and engage with closely related paralogs. Surprisingly, moonlighters and paralogs assembled co-translationally in only one but not all of the relevant assembly pathways. Our results highlight the regulatory complexity of assembly pathways. During the co-translational assembly of protein complexes, a fully synthesized subunit engages with the nascent chain of a newly synthesized interaction partner. Such events are thought to contribute to productive assembly, but their exact physiological relevance remains underexplored. Here, we examined structural motifs contained in nucleoporins for their potential to facilitate co-translational assembly. We experimentally tested candidate structural motifs and identified several previously unknown co-translational interactions. We demonstrate by selective ribosome profiling that domain invasion motifs of beta-propellers, coiled-coils, and short linear motifs act as co-translational assembly domains. Such motifs are often contained in proteins that are members of multiple complexes (moonlighters) and engage with closely related paralogs. Surprisingly, moonlighters and paralogs assembled co-translationally in only one but not all of the relevant assembly pathways. Our results highlight the regulatory complexity of assembly pathways.
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