ABSTRACT
Mitoribosomes are essential for the synthesis and maintenance of bioenergetic proteins. Here we use cryo-electron microscopy to determine a series of the small mitoribosomal subunit (SSU) intermediates in complex with auxiliary factors, revealing a sequential assembly mechanism. The methyltransferase TFB1M binds to partially unfolded rRNA h45 that is promoted by RBFA, while the mRNA channel is blocked. This enables binding of METTL15 that promotes further rRNA maturation and a large conformational change of RBFA. The new conformation allows initiation factor mtIF3 to already occupy the subunit interface during the assembly. Finally, the mitochondria-specific ribosomal protein mS37 (ref. 1) outcompetes RBFA to complete the assembly with the SSU-mS37-mtIF3 complex2 that proceeds towards mtIF2 binding and translation initiation. Our results explain how the action of step-specific factors modulate the dynamic assembly of the SSU, and adaptation of a unique protein, mS37, links the assembly to initiation to establish the catalytic human mitoribosome.
Subject(s)
Mitochondrial Ribosomes , Ribosome Subunits, Small , Humans , Cryoelectron Microscopy , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Eukaryotic Initiation Factors/chemistry , Eukaryotic Initiation Factors/metabolism , Mitochondria/chemistry , Mitochondria/metabolism , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/metabolism , Mitochondrial Ribosomes/chemistry , Mitochondrial Ribosomes/metabolism , Mitochondrial Ribosomes/ultrastructure , Ribosomal Proteins/chemistry , Ribosomal Proteins/metabolism , Ribosome Subunits, Small/chemistry , Ribosome Subunits, Small/metabolism , Ribosome Subunits, Small/ultrastructure , RNA, Ribosomal/chemistry , RNA, Ribosomal/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Transcription Factors/chemistry , Transcription Factors/metabolismABSTRACT
Altered expression of mitochondrial DNA (mtDNA) occurs in ageing and a range of human pathologies (for example, inborn errors of metabolism, neurodegeneration and cancer). Here we describe first-in-class specific inhibitors of mitochondrial transcription (IMTs) that target the human mitochondrial RNA polymerase (POLRMT), which is essential for biogenesis of the oxidative phosphorylation (OXPHOS) system1-6. The IMTs efficiently impair mtDNA transcription in a reconstituted recombinant system and cause a dose-dependent inhibition of mtDNA expression and OXPHOS in cell lines. To verify the cellular target, we performed exome sequencing of mutagenized cells and identified a cluster of amino acid substitutions in POLRMT that cause resistance to IMTs. We obtained a cryo-electron microscopy (cryo-EM) structure of POLRMT bound to an IMT, which further defined the allosteric binding site near the active centre cleft of POLRMT. The growth of cancer cells and the persistence of therapy-resistant cancer stem cells has previously been reported to depend on OXPHOS7-17, and we therefore investigated whether IMTs have anti-tumour effects. Four weeks of oral treatment with an IMT is well-tolerated in mice and does not cause OXPHOS dysfunction or toxicity in normal tissues, despite inducing a strong anti-tumour response in xenografts of human cancer cells. In summary, IMTs provide a potent and specific chemical biology tool to study the role of mtDNA expression in physiology and disease.
Subject(s)
Mitochondria/drug effects , Mitochondria/metabolism , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Transcription, Genetic/drug effects , Animals , Cell Proliferation/drug effects , Cryoelectron Microscopy , DNA, Mitochondrial/drug effects , DNA, Mitochondrial/genetics , DNA-Directed RNA Polymerases/metabolism , Down-Regulation/drug effects , Enzyme Stability/drug effects , Female , Gene Expression Regulation/drug effects , Genes, Mitochondrial/drug effects , Humans , Male , Mice , Neoplasms/drug therapy , Neoplasms/pathology , Substrate Specificity/drug effects , Xenograft Model Antitumor AssaysABSTRACT
ADP-ribosylation (ADPr) regulates important patho-physiological processes through its attachment to different amino acids in proteins. Recently, by precision mapping on all possible amino acid residues, we identified histone serine ADPr marks in the DNA damage response. However, the biochemical basis underlying this serine modification remained unknown. Here we report that serine ADPr is strictly dependent on histone PARylation factor 1 (HPF1), a recently identified regulator of PARP-1. Quantitative proteomics revealed that serine ADPr does not occur in cells lacking HPF1. Moreover, adding HPF1 to in vitro PARP-1/PARP-2 reactions is necessary and sufficient for serine-specific ADPr of histones and PARP-1 itself. Three endogenous serine ADPr sites are located on the PARP-1 automodification domain. Further identification of serine ADPr on HMG proteins and hundreds of other targets indicates that serine ADPr is a widespread modification. We propose that O-linked protein ADPr is the key signal in PARP-1/PARP-2-dependent processes that govern genome stability.
Subject(s)
Adenosine Diphosphate Ribose/metabolism , Carrier Proteins/metabolism , Histones/metabolism , Nuclear Proteins/metabolism , Poly (ADP-Ribose) Polymerase-1/metabolism , Poly(ADP-ribose) Polymerases/metabolism , Protein Processing, Post-Translational , Carrier Proteins/genetics , Cell Line, Tumor , Genomic Instability , Humans , Nuclear Proteins/genetics , Poly (ADP-Ribose) Polymerase-1/genetics , Poly(ADP-ribose) Polymerases/genetics , Proteomics/methods , Serine , TransfectionABSTRACT
The synthesis of mitochondrial OXPHOS complexes is central to cellular metabolism, yet many molecular details of mitochondrial translation remain elusive. It has been commonly held view that translation initiation in human mitochondria proceeded in a manner similar to bacterial systems, with the mitoribosomal small subunit bound to the initiation factors, mtIF2 and mtIF3, along with initiator tRNA and an mRNA. However, unlike in bacteria, most human mitochondrial mRNAs lack 5' leader sequences that can mediate small subunit binding, raising the question of how leaderless mRNAs are recognized by mitoribosomes. By using novel in vitro mitochondrial translation initiation assays, alongside biochemical and genetic characterization of cellular knockouts of mitochondrial translation factors, we describe unique features of translation initiation in human mitochondria. We show that in vitro, leaderless mRNA transcripts can be loaded directly onto assembled 55S mitoribosomes, but not onto the mitoribosomal small subunit (28S), in a manner that requires initiator fMet-tRNAMet binding. In addition, we demonstrate that in human cells and in vitro, mtIF3 activity is not required for translation of leaderless mitochondrial transcripts but is essential for translation of ATP6 in the case of the bicistronic ATP8/ATP6 transcript. Furthermore, we show that mtIF2 is indispensable for mitochondrial protein synthesis. Our results demonstrate an important evolutionary divergence of the mitochondrial translation system and further our fundamental understanding of a process central to eukaryotic metabolism.
Subject(s)
Mitochondria , Peptide Chain Initiation, Translational , Animals , Humans , Bacteria/genetics , Mammals/genetics , Mitochondria/physiology , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Peptide Initiation Factors/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
Drosophila melanogaster has been a workhorse of genetics and cell biology for more than a century. However, proteomic-based methods have been limited due to the complexity and dynamic range of the fly proteome and the lack of efficient labeling methods. Here, we advanced a chemically defined food source into direct stable-isotope labeling of amino acids in flies (SILAF). It allows for the rapid and cost-efficient generation of a large number of larvae or flies, with full incorporation of lysine-[13C6] after six labeling days. SILAF followed by fractionation and enrichment gave proteomic insights at a depth of 7196 proteins and 8451 phosphorylation sites, which substantiated metabolic regulation on enzymatic level. We applied SILAF to quantify the mitochondrial phosphoproteome of an early-stage leucine-rich PPR motif-containing protein (LRPPRC)-knockdown fly model of mitochondrial disease that almost exclusively affects protein levels of the oxidative phosphorylation (OXPHOS) system. While the mitochondrial compartment was hypo-phosphorylated, two conserved phosphosites on OXPHOS subunits NDUFB10 and NDUFA4 were significantly upregulated upon impaired OXPHOS function. The ease and versatility of the method actuate the fruit fly as an appealing model in proteomic and posttranslational modification studies, and it enlarges potential metabolic applications based on heavy amino acid diets.
Subject(s)
Drosophila Proteins/metabolism , Mitochondrial Proteins/metabolism , Phosphoproteins/metabolism , Amino Acids/metabolism , Animals , Drosophila melanogaster , Female , Isotope Labeling , Male , Phosphorylation , ProteomeABSTRACT
Human mitoribosomes are macromolecular complexes essential for translation of 11 mitochondrial mRNAs. The large and the small mitoribosomal subunits undergo a multistep maturation process that requires the involvement of several factors. Among these factors, GTP-binding proteins (GTPBPs) play an important role as GTP hydrolysis can provide energy throughout the assembly stages. In bacteria, many GTPBPs are needed for the maturation of ribosome subunits and, of particular interest for this study, ObgE has been shown to assist in the 50S subunit assembly. Here, we characterize the role of a related human Obg-family member, GTPBP5. We show that GTPBP5 interacts specifically with the large mitoribosomal subunit (mt-LSU) proteins and several late-stage mitoribosome assembly factors, including MTERF4:NSUN4 complex, MRM2 methyltransferase, MALSU1 and MTG1. Interestingly, we find that interaction of GTPBP5 with the mt-LSU is compromised in the presence of a non-hydrolysable analogue of GTP, implying a different mechanism of action of this protein in contrast to that of other Obg-family GTPBPs. GTPBP5 ablation leads to severe impairment in the oxidative phosphorylation system, concurrent with a decrease in mitochondrial translation and reduced monosome formation. Overall, our data indicate an important role of GTPBP5 in mitochondrial function and suggest its involvement in the late-stage of mt-LSU maturation.
Subject(s)
Mitochondrial Proteins/metabolism , Mitochondrial Ribosomes/metabolism , Monomeric GTP-Binding Proteins/physiology , Ribosomal Proteins/metabolism , Ribosome Subunits, Large, Eukaryotic/metabolism , Bone Neoplasms/pathology , CRISPR-Cas Systems , Cell Line, Tumor , Gene Expression Regulation , Gene Knockout Techniques , Guanosine Triphosphate/metabolism , HEK293 Cells , Humans , Osteosarcoma/pathology , Oxidative Phosphorylation , Protein Interaction MappingABSTRACT
Regulation of replication and expression of mitochondrial DNA (mtDNA) is essential for cellular energy conversion via oxidative phosphorylation. The mitochondrial transcription elongation factor (TEFM) has been proposed to regulate the switch between transcription termination for replication primer formation and processive, near genome-length transcription for mtDNA gene expression. Here, we report that Tefm is essential for mouse embryogenesis and that levels of promoter-distal mitochondrial transcripts are drastically reduced in conditional Tefm-knockout hearts. In contrast, the promoter-proximal transcripts are much increased in Tefm knockout mice, but they mostly terminate before the region where the switch from transcription to replication occurs, and consequently, de novo mtDNA replication is profoundly reduced. Unexpectedly, deep sequencing of RNA from Tefm knockouts revealed accumulation of unprocessed transcripts in addition to defective transcription elongation. Furthermore, a proximity-labeling (BioID) assay showed that TEFM interacts with multiple RNA processing factors. Our data demonstrate that TEFM acts as a general transcription elongation factor, necessary for both gene transcription and replication primer formation, and loss of TEFM affects RNA processing in mammalian mitochondria.
Subject(s)
Mitochondria/genetics , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , RNA Processing, Post-Transcriptional , Transcription Elongation, Genetic , Transcription Factors/metabolism , Animals , DNA, Mitochondrial , Embryonic Development/genetics , Gene Deletion , Gene Expression Regulation , Genetic Loci , Heterozygote , Mice , Mice, Knockout , Mitochondria/ultrastructure , Phenotype , Promoter Regions, GeneticABSTRACT
In all biological systems, RNAs are associated with RNA-binding proteins (RBPs), forming complexes that control gene regulatory mechanisms, from RNA synthesis to decay. In mammalian mitochondria, post-transcriptional regulation of gene expression is conducted by mitochondrial RBPs (mt-RBPs) at various stages of mt-RNA metabolism, including polycistronic transcript production, its processing into individual transcripts, mt-RNA modifications, stability, translation and degradation. To date, only a handful of mt-RBPs have been characterized. Here, we describe a putative human mitochondrial protein, C6orf203, that contains an S4-like domain-an evolutionarily conserved RNA-binding domain previously identified in proteins involved in translation. Our data show C6orf203 to bind highly structured RNA in vitro and associate with the mitoribosomal large subunit in HEK293T cells. Knockout of C6orf203 leads to a decrease in mitochondrial translation and consequent OXPHOS deficiency, without affecting mitochondrial RNA levels. Although mitoribosome stability is not affected in C6orf203-depleted cells, mitoribosome profiling analysis revealed a global disruption of the association of mt-mRNAs with the mitoribosome, suggesting that C6orf203 may be required for the proper maturation and functioning of the mitoribosome. We therefore propose C6orf203 to be a novel RNA-binding protein involved in mitochondrial translation, expanding the repertoire of factors engaged in this process.
Subject(s)
Mitochondria/genetics , Mitochondrial Proteins/biosynthesis , RNA, Mitochondrial/genetics , RNA-Binding Proteins/genetics , Animals , HEK293 Cells , Humans , Mitochondrial Proteins/genetics , Mitochondrial Proteins/physiology , Mitochondrial Ribosomes/metabolism , RNA, Messenger/genetics , RNA, Ribosomal/genetics , RNA-Binding Proteins/physiologyABSTRACT
BACKGROUND: RNA-binding proteins (RBPs) are fundamental regulators of cellular biology that affect all steps in the generation and processing of RNA molecules. Recent evidence suggests that regulation of RBPs that modulate both RNA stability and translation may have a profound effect on the proteome. However, regulation of RBPs in clinically relevant experimental conditions has not been studied systematically. METHODS: We used RNA interactome capture, a method for the global identification of RBPs to characterize the global RNA-binding proteome (RBPome) associated with polyA-tailed RNA species in murine ciliated epithelial cells of the inner medullary collecting duct. To study regulation of RBPs in a clinically relevant condition, we analyzed hypoxia-associated changes of the RBPome. RESULTS: We identified >1000 RBPs that had been previously found using other systems. In addition, we found a number of novel RBPs not identified by previous screens using mouse or human cells, suggesting that these proteins may be specific RBPs in differentiated kidney epithelial cells. We also found quantitative differences in RBP-binding to mRNA that were associated with hypoxia versus normoxia. CONCLUSIONS: These findings demonstrate the regulation of RBPs through environmental stimuli and provide insight into the biology of hypoxia-response signaling in epithelial cells in the kidney. A repository of the RBPome and proteome in kidney tubular epithelial cells, derived from our findings, is freely accessible online, and may contribute to a better understanding of the role of RNA-protein interactions in kidney tubular epithelial cells, including the response of these cells to hypoxia.
Subject(s)
Epithelial Cells/metabolism , Kidney Tubules, Collecting/cytology , Kidney Tubules, Collecting/metabolism , Proteome/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Animals , Cell Differentiation , Cell Hypoxia/physiology , Cilia/metabolism , HEK293 Cells , Humans , Mice , Protein BindingABSTRACT
Ribosome profiling via high-throughput sequencing (ribo-seq) is a promising new technique for characterizing the occupancy of ribosomes on messenger RNA (mRNA) at base-pair resolution. The ribosome is responsible for translating mRNA into proteins, so information about its occupancy offers a detailed view of ribosome density and position which could be used to discover new translated open reading frames (ORFs), among other things. In this work, we propose Rp-Bp, an unsupervised Bayesian approach to predict translated ORFs from ribosome profiles. We use state-of-the-art Markov chain Monte Carlo techniques to estimate posterior distributions of the likelihood of translation of each ORF. Hence, an important feature of Rp-Bp is its ability to incorporate and propagate uncertainty in the prediction process. A second novel contribution is automatic Bayesian selection of read lengths and ribosome P-site offsets (BPPS). We empirically demonstrate that our read length selection technique modestly improves sensitivity by identifying more canonical and non-canonical ORFs. Proteomics- and quantitative translation initiation sequencing-based validation verifies the high quality of all of the predictions. Experimental comparison shows that Rp-Bp results in more peptide identifications and proteomics-validated ORF predictions compared to another recent tool for translation prediction.
Subject(s)
High-Throughput Nucleotide Sequencing/methods , Protein Biosynthesis , Ribosomes/chemistry , Sequence Analysis, RNA/methods , Animals , Bayes Theorem , Caenorhabditis elegans/genetics , Computer Simulation , HEK293 Cells , Humans , Mice , Open Reading Frames , Peptides/chemistry , Proteomics , Ribosomes/metabolismABSTRACT
ADP-ribosylation (ADPr) is a biologically and clinically important post-translational modification, but little is known about the amino acids it targets on cellular proteins. Here we present a proteomic approach for direct in vivo identification and quantification of ADPr sites on histones. We have identified 12 unique ADPr sites in human osteosarcoma cells and report serine ADPr as a new type of histone mark that responds to DNA damage.
Subject(s)
Adenosine Diphosphate/metabolism , Histones/chemistry , Histones/metabolism , Protein Processing, Post-Translational , Serine/metabolism , Cell Line, Tumor , DNA Damage , Humans , ProteomicsABSTRACT
LRPPRC is a protein that has attracted interest both for its role in post-transcriptional regulation of mitochondrial gene expression and more recently because numerous mutated variants have been characterized as causing severe infantile mitochondrial neurodegeneration. LRPPRC belongs to the pentatricopeptide repeat (PPR) protein family, originally defined by their RNA binding capacity, and forms a complex with SLIRP that harbours an RNA recognition motif (RRM) domain. We show here that LRPPRC displays a broad and strong RNA binding capacity in vitro in contrast to SLIRP that associates only weakly with RNA. The LRPPRC-SLIRP complex comprises a hetero-dimer via interactions by polar amino acids in the single RRM domain of SLIRP and three neighbouring PPR motifs in the second quarter of LRPPRC, which critically contribute to the LRPPRC-SLIRP binding interface to enhance its stability. Unexpectedly, specific amino acids at this interface are located within the PPRs of LRPPRC at positions predicted to interact with RNA and within the RNP1 motif of SLIRP's RRM domain. Our findings thus unexpectedly establish that despite the prediction that these residues in LRPPRC and SLIRP should bind RNA, they are instead used to facilitate protein-protein interactions, enabling the formation of a stable complex between these two proteins.
Subject(s)
Neoplasm Proteins/metabolism , RNA Recognition Motif , RNA-Binding Proteins/metabolism , Repetitive Sequences, Amino Acid , Amino Acid Sequence , Amino Acids/genetics , Conserved Sequence , Cross-Linking Reagents/metabolism , HEK293 Cells , Humans , Models, Biological , Mutation/genetics , Neoplasm Proteins/chemistry , Protein Binding , Protein Multimerization , Protein Stability , RNA/metabolism , RNA-Binding Proteins/chemistryABSTRACT
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 important 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 focus on exploring the function of GTPBP8, the human homolog of EngB. We find that ablation of GTPBP8 leads to the inhibition of mitochondrial translation, resulting in significant impairment of oxidative phosphorylation. Structural analysis of mitoribosomes from GTPBP8 knock-out cells shows the accumulation of mitoribosomal large subunit assembly intermediates that are incapable of forming functional monosomes. Furthermore, fPAR-CLIP analysis reveals that GTPBP8 is an RNA-binding protein that interacts specifically with the mitochondrial ribosome large subunit 16 S rRNA. Our study highlights the role of GTPBP8 as a component of the mitochondrial gene expression machinery involved in mitochondrial large subunit maturation.
Subject(s)
GTP-Binding Proteins , Mitochondria , Mitochondrial Ribosomes , Oxidative Phosphorylation , Humans , Mitochondrial Ribosomes/metabolism , Mitochondria/metabolism , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/genetics , RNA, Ribosomal, 16S/genetics , RNA, Ribosomal, 16S/metabolism , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Mitochondrial Proteins/metabolism , Mitochondrial Proteins/genetics , HEK293 Cells , Protein Biosynthesis , RNA, Messenger/metabolism , RNA, Messenger/genetics , HeLa CellsABSTRACT
The in-depth analysis of complex proteome samples requires fractionation of the sample into subsamples prior to LC-MS/MS in shotgun proteomics experiments. We have established a 3D workflow for shotgun proteomics that relies on protein separation by 1D PAGE, gel fractionation, trypsin digestion, and peptide separation by in-gel IEF, prior to RP-HPLC-MS/MS. Our results show that applying peptide IEF can significantly increase the number of proteins identified from PAGE subfractionation. This method delivers deeper proteome coverage and provides a large degree of flexibility in experimentally approaching highly complex mixtures by still relying on protein separation according to molecular weight in the first dimension.
Subject(s)
Electrophoresis, Polyacrylamide Gel/methods , Isoelectric Focusing/methods , Peptides/isolation & purification , Proteome/metabolism , Proteomics/methods , Chromatography, Liquid , HeLa Cells , Humans , Mass Spectrometry , Reproducibility of Results , Trypsin/metabolismABSTRACT
Mitochondrial dysfunction and cellular senescence are hallmarks of aging. However, the relationship between these two phenomena remains incompletely understood. In this study, we investigated the rewiring of mitochondria upon development of the senescent state in human IMR90 fibroblasts. Determining the bioenergetic activities and abundance of mitochondria, we demonstrate that senescent cells accumulate mitochondria with reduced OXPHOS activity, resulting in an overall increase of mitochondrial activities in senescent cells. Time-resolved proteomic analyses revealed extensive reprogramming of the mitochondrial proteome upon senescence development and allowed the identification of metabolic pathways that are rewired with different kinetics upon establishment of the senescent state. Among the early responding pathways, the degradation of branched-chain amino acid was increased, whereas the one carbon folate metabolism was decreased. Late-responding pathways include lipid metabolism and mitochondrial translation. These signatures were confirmed by metabolic flux analyses, highlighting metabolic rewiring as a central feature of mitochondria in cellular senescence. Together, our data provide a comprehensive view on the changes in mitochondrial proteome in senescent cells and reveal how the mitochondrial metabolism is rewired in senescent cells.
Subject(s)
Proteome , Proteomics , Humans , Proteome/metabolism , Mitochondria/metabolism , Aging/metabolism , Cellular SenescenceABSTRACT
The mitochondrial translation machinery highly diverged from its bacterial counterpart. This includes deviation from the universal genetic code, with AGA and AGG codons lacking cognate tRNAs in human mitochondria. The locations of these codons at the end of COX1 and ND6 open reading frames, respectively, suggest they might function as stop codons. However, while the canonical stop codons UAA and UAG are known to be recognized by mtRF1a, the release mechanism at AGA and AGG codons remains a debated issue. Here, we show that upon the loss of another member of the mitochondrial release factor family, mtRF1, mitoribosomes accumulate specifically at AGA and AGG codons. Stalling of mitoribosomes alters COX1 transcript and protein levels, but not ND6 synthesis. In addition, using an in vitro reconstituted mitochondrial translation system, we demonstrate the specific peptide release activity of mtRF1 at the AGA and AGG codons. Together, our results reveal the role of mtRF1 in translation termination at non-canonical stop codons in mitochondria.
Subject(s)
Codon, Terminator , Mitochondria , Peptide Termination Factors , Humans , Codon, Terminator/metabolism , Mitochondria/metabolism , Peptide Termination Factors/metabolism , Protein Biosynthesis , Ribosomes/metabolismABSTRACT
The mammalian respiratory chain complexes I, III2, and IV (CI, CIII2, and CIV) are critical for cellular bioenergetics and form a stable assembly, the respirasome (CI-CIII2-CIV), that is biochemically and structurally well documented. The role of the respirasome in bioenergetics and the regulation of metabolism is subject to intense debate and is difficult to study because the individual respiratory chain complexes coexist together with high levels of respirasomes. To critically investigate the in vivo role of the respirasome, we generated homozygous knockin mice that have normal levels of respiratory chain complexes but profoundly decreased levels of respirasomes. Surprisingly, the mutant mice are healthy, with preserved respiratory chain capacity and normal exercise performance. Our findings show that high levels of respirasomes are dispensable for maintaining bioenergetics and physiology in mice but raise questions about their alternate functions, such as those relating to the regulation of protein stability and prevention of age-associated protein aggregation.
Subject(s)
Mitochondria , Mitochondrial Membranes , Animals , Mice , Electron Transport , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Electron Transport Complex IV/metabolism , Energy Metabolism , Mammals/metabolismABSTRACT
The outer mitochondrial membrane (OMM) is essential for cellular homeostasis. Yet little is known of the mechanisms that remodel it during natural stresses. We found that large "SPOTs" (structures positive for OMM) emerge during Toxoplasma gondii infection in mammalian cells. SPOTs mediated the depletion of the OMM proteins mitofusin 1 and 2, which restrict parasite growth. The formation of SPOTs depended on the parasite effector TgMAF1 and the host mitochondrial import receptor TOM70, which is required for optimal parasite proliferation. TOM70 enabled TgMAF1 to interact with the host OMM translocase SAM50. The ablation of SAM50 or the overexpression of an OMM-targeted protein promoted OMM remodeling independently of infection. Thus, Toxoplasma hijacks the formation of SPOTs, a cellular response to OMM stress, to promote its growth.
Subject(s)
Mitochondrial Membranes/physiology , Mitochondrial Precursor Protein Import Complex Proteins/metabolism , Protozoan Proteins/metabolism , Toxoplasma/physiology , Animals , Cell Line , GTP Phosphohydrolases/metabolism , Humans , Intracellular Membranes/physiology , Intracellular Membranes/ultrastructure , Mice , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Membranes/ultrastructure , Mitochondrial Proteins/metabolism , Protein Binding , Stress, Physiological , Toxoplasma/growth & development , Toxoplasma/ultrastructure , Toxoplasmosis/parasitology , Vacuoles/physiology , Vacuoles/ultrastructureABSTRACT
Nowadays identification and quantification of proteins from biological samples by mass spectrometry are widely used. For the identification of proteins, there are two scenarios. Proteins are either pre-fractionated in some way, e.g., by gel electrophoresis or chromatography, or analyzed as complex mixture (shotgun). Because of technological developments of mass spectrometry, the identification of several thousand proteins from complex biological matrix becomes possible. However, in many cases, it is still useful to separate proteins first in a gel. For quantifying proteins, label-free, isotopic labeling, and data-independent acquisition (DIA) library are widely used. Not only mass spectrometry technology made progress. This is also true for the sample preparation. Protocols and techniques developed recently not only make the analysis of starting material in the low microgram range possible but also simplify the whole procedure. Here, we will describe some detailed protocols of preparing samples for mass spectrometry-based protein identification and protein quantification, as in-gel digestion, in-solution digestion, peptide cleaning, and TMT labeling. This will allow also inexperienced beginners to get good results.
Subject(s)
Analytic Sample Preparation Methods , Mass Spectrometry , Proteins/analysis , Proteomics , Centrifugation , Electrophoresis, Gel, Two-Dimensional , Filtration , ProteolysisABSTRACT
Protein-focused research has been challenging in Drosophila melanogaster due to few specific antibodies for Western blotting and the lack of effective labeling methods for quantitative proteomics. Herein, we describe the preparation of a holidic medium that allows stable-isotope labeling of amino acids in fruit flies (SILAF). Furthermore, in this chapter, we provide a protocol for mitochondrial enrichments from Drosophila larvae and flies together with a procedure to generate high-quality peptides for further analysis by mass spectrometry. Samples obtained following this protocol can be used for various functional studies such as comprehensive proteome profiling or quantitative analysis of posttranslational modifications upon enrichment. SILAF is based on standard fly routines in a basic wet lab environment and provides a flexible and cost-effective tool for quantitative protein expression analysis.