ABSTRACT
The mitochondrial proteome is built mainly by import of nuclear-encoded precursors, which are targeted mostly by cleavable presequences. Presequence processing upon import is essential for proteostasis and survival, but the consequences of dysfunctional protein maturation are unknown. We find that impaired presequence processing causes accumulation of precursors inside mitochondria that form aggregates, which escape degradation and unexpectedly do not cause cell death. Instead, cells survive via activation of a mitochondrial unfolded protein response (mtUPR)-like pathway that is triggered very early after precursor accumulation. In contrast to classical stress pathways, this immediate response maintains mitochondrial protein import, membrane potential, and translation through translocation of the nuclear HMG-box transcription factor Rox1 to mitochondria. Rox1 binds mtDNA and performs a TFAM-like function pivotal for transcription and translation. Induction of early mtUPR provides a reversible stress model to mechanistically dissect the initial steps in mtUPR pathways with the stressTFAM Rox1 as the first line of defense.
Subject(s)
Mitochondria/metabolism , Repressor Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Transcription Factors/metabolism , Unfolded Protein Response/physiology , Cell Death/physiology , Cell Nucleus/metabolism , DNA, Mitochondrial/metabolism , Membrane Potentials/physiology , Protein Biosynthesis/physiology , Saccharomyces cerevisiae/metabolism , Transcription, Genetic/physiologyABSTRACT
Mitochondria import a large number of nuclear-encoded proteins via membrane-bound transport machineries; however, little is known about regulation of the preprotein translocases. We report that the main protein entry gate of mitochondria, the translocase of the outer membrane (TOM complex), is phosphorylated by cytosolic kinases-in particular, casein kinase 2 (CK2) and protein kinase A (PKA). CK2 promotes biogenesis of the TOM complex by phosphorylation of two key components, the receptor Tom22 and the import protein Mim1, which in turn are required for import of further Tom proteins. Inactivation of CK2 decreases the levels of the TOM complex and thus mitochondrial protein import. PKA phosphorylates Tom70 under nonrespiring conditions, thereby inhibiting its receptor activity and the import of mitochondrial metabolite carriers. We conclude that cytosolic kinases exert stimulatory and inhibitory effects on biogenesis and function of the TOM complex and thus regulate protein import into mitochondria.
Subject(s)
Casein Kinase II/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Cytosol/enzymology , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Carrier Proteins/metabolism , Cytosol/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Precursor Protein Import Complex Proteins , Phosphorylation , Protein Transport , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Membrane proteins require protein machineries to insert their hydrophobic transmembrane domains (TMDs) into the lipid bilayer. A functional analysis of protein insertases in this issue of PLOS Biology reveals that the fundamental mechanism of membrane protein insertion is universally conserved.
Subject(s)
Lipid Bilayers , Membrane Proteins , Hydrophobic and Hydrophilic Interactions , Lipid Bilayers/chemistry , Membrane Proteins/genetics , Membrane Proteins/metabolismABSTRACT
Many mitochondrial proteins are synthesized with N-terminal presequences that are removed by specific peptidases. The N-termini of the mature proteins and thus peptidase cleavage sites have only been determined for a small fraction of mitochondrial proteins and yielded a controversial situation for the cleavage site specificity of the major mitochondrial processing peptidase (MPP). We report a global analysis of the N-proteome of yeast mitochondria, revealing the N-termini of 615 different proteins. Significantly more proteins than predicted contained cleavable presequences. We identified the intermediate cleaving peptidase Icp55, which removes an amino acid from a characteristic set of MPP-generated N-termini, solving the controversial situation of MPP specificity and suggesting that Icp55 converts instable intermediates into stable proteins. Our results suggest that Icp55 is critical for stabilization of the mitochondrial proteome and illustrate how the N-proteome can serve as rich source for a systematic analysis of mitochondrial protein targeting, cleavage and turnover.
Subject(s)
Mitochondria/chemistry , Mitochondrial Proteins/analysis , Proteome/analysis , Saccharomyces cerevisiae/chemistry , Humans , Peptide Hydrolases/metabolism , Protein StabilityABSTRACT
Zellweger spectrum disorder (ZSD) is the most severe peroxisomal biogenesis disorder (PBD). Why ZSD patients not only loose functional peroxisomes but also present with severe mitochondrial dysfunction was a long-standing mystery. In this issue, Nuebel et al (2021) identified that loss of peroxisomes leads to re-routing of peroxisomal proteins to mitochondria, thereby impairing mitochondrial structure and function. The findings provide the first molecular understanding of the mitochondrial-peroxisomal link in ZSD.
Subject(s)
Peroxisomal Disorders , Zellweger Syndrome , Humans , Mitochondria , Peroxins/metabolism , Peroxisomal Disorders/metabolism , Peroxisomes/metabolism , Zellweger Syndrome/metabolismABSTRACT
Mitochondria are central to cellular energetics, metabolism, and signaling. In this issue, Pagliarini et al. (2008) report the largest compendium of mammalian mitochondrial proteins to date. Together with proteomic studies in yeast, this study represents an important step toward the systematic characterization of the mitochondrial proteome and of mitochondrial diseases.
Subject(s)
Mitochondria/chemistry , Mitochondrial Proteins/analysis , Proteome , Animals , Humans , Mass Spectrometry , MiceABSTRACT
Communication of mitochondria with the rest of the cell requires beta-barrel proteins of the outer membrane. All beta-barrel proteins are synthesized as precursors in the cytosol and imported into mitochondria by the general translocase TOM and the sorting machinery SAM. The SAM complex contains two proteins essential for cell viability, the channel-forming Sam50 and Sam35. We have identified the sorting signal of mitochondrial beta-barrel proteins that is universal in all eukaryotic kingdoms. The beta-signal initiates precursor insertion into a hydrophilic, proteinaceous membrane environment by forming a ternary complex with Sam35 and Sam50. Sam35 recognizes the beta-signal, inducing a major conductance increase of the Sam50 channel. Subsequent precursor release from SAM is coupled to integration into the lipid phase. We propose that a two-stage mechanism of signal-driven insertion into a membrane protein complex and subsequent integration into the lipid phase may represent a general mechanism for biogenesis of beta-barrel proteins.
Subject(s)
Mitochondrial Membranes/metabolism , Mitochondrial Proteins/metabolism , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/metabolism , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins , Mitochondrial Membranes/chemistry , Mitochondrial Proteins/chemistry , Protein Sorting Signals , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Transport , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Mitochondria contain approximately 1,000 different proteins, most of which are imported from the cytosol. Two import pathways that direct proteins into the mitochondrial inner membrane and matrix have been known for many years. The identification of numerous new transport components in recent proteomic studies has led to novel mechanistic insight into these pathways and the discovery of new import pathways into the outer membrane and intermembrane space. Protein translocases do not function as independent units but are integrated into dynamic networks and are connected to machineries that function in bioenergetics, mitochondrial morphology and coupling to the endoplasmic reticulum.
Subject(s)
Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Proteins/metabolism , Proteomics , Animals , Humans , Mitochondrial Membranes/metabolism , Protein TransportABSTRACT
An experimental and computational approach for identification of protein-protein interactions by ex vivo chemical crosslinking and mass spectrometry (CLMS) has been developed that takes advantage of the specific characteristics of cyanurbiotindipropionylsuccinimide (CBDPS), an affinity-tagged isotopically coded mass spectrometry (MS)-cleavable crosslinking reagent. Utilizing this reagent in combination with a crosslinker-specific data-dependent acquisition strategy based on MS2 scans, and a software pipeline designed for integrating crosslinker-specific mass spectral information led to demonstrated improvements in the application of the CLMS technique, in terms of the detection, acquisition, and identification of crosslinker-modified peptides. This approach was evaluated on intact yeast mitochondria, and the results showed that hundreds of unique protein-protein interactions could be identified on an organelle proteome-wide scale. Both known and previously unknown protein-protein interactions were identified. These interactions were assessed based on their known sub-compartmental localizations. Additionally, the identified crosslinking distance constraints are in good agreement with existing structural models of protein complexes involved in the mitochondrial electron transport chain.
Subject(s)
Cross-Linking Reagents/chemistry , Isotope Labeling , Mass Spectrometry , Organelles/metabolism , Protein Interaction Mapping/methods , Biotin/analogs & derivatives , Chemical Fractionation , Mitochondria/metabolism , Models, Molecular , Peptides/metabolism , Protein Interaction Maps , Saccharomyces cerevisiae/metabolism , SuccinimidesABSTRACT
Mitochondria cannot form de novo but require mechanisms that mediate their inheritance to daughter cells. The parasitic protozoan Trypanosoma brucei has a single mitochondrion with a single-unit genome that is physically connected across the two mitochondrial membranes with the basal body of the flagellum. This connection, termed the tripartite attachment complex (TAC), is essential for the segregation of the replicated mitochondrial genomes prior to cytokinesis. Here we identify a protein complex consisting of three integral mitochondrial outer membrane proteins-TAC60, TAC42 and TAC40-which are essential subunits of the TAC. TAC60 contains separable mitochondrial import and TAC-sorting signals and its biogenesis depends on the main outer membrane protein translocase. TAC40 is a member of the mitochondrial porin family, whereas TAC42 represents a novel class of mitochondrial outer membrane ß-barrel proteins. Consequently TAC40 and TAC42 contain C-terminal ß-signals. Thus in trypanosomes the highly conserved ß-barrel protein assembly machinery plays a major role in the biogenesis of its unique mitochondrial genome segregation system.
Subject(s)
DNA, Kinetoplast/biosynthesis , DNA, Kinetoplast/genetics , DNA, Mitochondrial/biosynthesis , DNA, Mitochondrial/genetics , Trypanosoma brucei brucei/genetics , Trypanosoma brucei brucei/metabolism , Animals , Genome, Mitochondrial , Genome, Protozoan , Humans , Mitochondrial Dynamics , Mitochondrial Membranes/metabolism , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Protein Sorting Signals/genetics , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Trypanosoma brucei brucei/pathogenicityABSTRACT
The biochemical analysis of protein phosphorylation in mitochondria lags behind that of cytosolic signaling events. One reason is the poor stability of many phosphorylation sites during common isolation procedures for mitochondria. We present here an optimized, fast protocol for the purification of yeast mitochondria that greatly increases recovery of phosphorylated mitochondrial proteins. Moreover, we describe improved protocols for the biochemical analysis of mitochondrial protein phosphorylation by Zn2+-Phos-tag electrophoresis under both denaturing and - for the first time - native conditions, and demonstrate that they outperform previously applied methods.
Subject(s)
Mitochondrial Proteins/metabolism , Phosphoproteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Electrophoresis, Polyacrylamide Gel/methods , Mitochondria/metabolism , Mitochondrial Proteins/isolation & purification , Phosphoproteins/isolation & purification , Phosphorylation , Pyridines , Saccharomyces cerevisiae Proteins/isolation & purification , ZincABSTRACT
Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) govern cellular homeostasis by inducing signaling. H2O2 modulates the activity of phosphatases and many other signaling molecules through oxidation of critical cysteine residues, which led to the notion that initiation of ROS signaling is broad and nonspecific, and thus fundamentally distinct from other signaling pathways. Here, we report that H2O2 signaling bears hallmarks of a regular signal transduction cascade. It is controlled by hierarchical signaling events resulting in a focused response as the results place the mitochondrial respiratory chain upstream of tyrosine-protein kinase Lyn, Lyn upstream of tyrosine-protein kinase SYK (Syk), and Syk upstream of numerous targets involved in signaling, transcription, translation, metabolism, and cell cycle regulation. The active mediators of H2O2 signaling colocalize as H2O2 induces mitochondria-associated Lyn and Syk phosphorylation, and a pool of Lyn and Syk reside in the mitochondrial intermembrane space. Finally, the same intermediaries control the signaling response in tissues and species responsive to H2O2 as the respiratory chain, Lyn, and Syk were similarly required for H2O2 signaling in mouse B cells, fibroblasts, and chicken DT40 B cells. Consistent with a broad role, the Syk pathway is coexpressed across tissues, is of early metazoan origin, and displays evidence of evolutionary constraint in the human. These results suggest that H2O2 signaling is under control of a signal transduction pathway that links the respiratory chain to the mitochondrial intermembrane space-localized, ubiquitous, and ancient Syk pathway in hematopoietic and nonhematopoietic cells.
Subject(s)
Electron Transport , Hydrogen Peroxide/metabolism , Mitochondrial Membranes/metabolism , Signal Transduction , Animals , Cells, Cultured , Chickens , Enzyme Activation , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Phosphorylation , Protein-Tyrosine Kinases/metabolism , Reactive Oxygen Species/metabolism , Syk Kinase , Tyrosine/metabolismABSTRACT
Mitochondria are essential for eukaryotic life and more than 95% of their proteins are imported as precursors from the cytosol. The targeting signals for this posttranslational import are conserved in all eukaryotes. However, this conservation does not hold true for the protein translocase of the mitochondrial outer membrane that serves as entry gate for essentially all precursor proteins. Only two of its subunits, Tom40 and Tom22, are conserved and thus likely were present in the last eukaryotic common ancestor. Tom7 is found in representatives of all supergroups except the Excavates. This suggests that it was added to the core of the translocase after the Excavates segregated from all other eukaryotes. A comparative analysis of the biochemically and functionally characterized outer membrane translocases of yeast, plants, and trypanosomes, which represent three eukaryotic supergroups, shows that the receptors that recognize the conserved import signals differ strongly between the different systems. They present a remarkable example of convergent evolution at the molecular level. The structural diversity of the functionally conserved import receptors therefore provides insight into the early evolutionary history of mitochondria.
Subject(s)
Biological Evolution , Eukaryota/genetics , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Eukaryota/classification , Mitochondrial Membrane Transport Proteins/chemistry , Mitochondrial Proteins/metabolism , Multigene Family , Multiprotein Complexes , Plants/metabolism , Porins/chemistry , Porins/metabolism , Protein Binding , Protein Subunits/metabolism , Protein Transport , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/metabolism , Symbiosis , Trypanosoma/metabolism , Yeasts/metabolismABSTRACT
Biogenesis of complex IV of the mitochondrial respiratory chain requires assembly factors for subunit maturation, co-factor attachment and stabilization of intermediate assemblies. A pathogenic mutation in COA6, leading to substitution of a conserved tryptophan for a cysteine residue, results in a loss of complex IV activity and cardiomyopathy. Here, we demonstrate that the complex IV defect correlates with a severe loss in complex IV assembly in patient heart but not fibroblasts. Complete loss of COA6 activity using gene editing in HEK293T cells resulted in a profound growth defect due to complex IV deficiency, caused by impaired biogenesis of the copper-bound mitochondrial DNA-encoded subunit COX2 and subsequent accumulation of complex IV assembly intermediates. We show that the pathogenic mutation in COA6 does not affect its import into mitochondria but impairs its maturation and stability. Furthermore, we show that COA6 has the capacity to bind copper and can associate with newly translated COX2 and the mitochondrial copper chaperone SCO1. Our data reveal that COA6 is intricately involved in the copper-dependent biogenesis of COX2.
Subject(s)
Cardiomyopathies/genetics , Carrier Proteins/genetics , Electron Transport Complex IV/genetics , Membrane Proteins/metabolism , Mitochondrial Proteins/genetics , Carrier Proteins/metabolism , Copper/metabolism , Electron Transport Complex IV/metabolism , Fibroblasts/cytology , Fibroblasts/enzymology , HEK293 Cells , Humans , Infant , Male , Mitochondrial Proteins/metabolism , Molecular ChaperonesABSTRACT
Malfunctioning of the protein α-synuclein is critically involved in the demise of dopaminergic neurons relevant to Parkinson's disease. Nonetheless, the precise mechanisms explaining this pathogenic neuronal cell death remain elusive. Endonuclease G (EndoG) is a mitochondrially localized nuclease that triggers DNA degradation and cell death upon translocation from mitochondria to the nucleus. Here, we show that EndoG displays cytotoxic nuclear localization in dopaminergic neurons of human Parkinson-diseased patients, while EndoG depletion largely reduces α-synuclein-induced cell death in human neuroblastoma cells. Xenogenic expression of human α-synuclein in yeast cells triggers mitochondria-nuclear translocation of EndoG and EndoG-mediated DNA degradation through a mechanism that requires a functional kynurenine pathway and the permeability transition pore. In nematodes and flies, EndoG is essential for the α-synuclein-driven degeneration of dopaminergic neurons. Moreover, the locomotion and survival of α-synuclein-expressing flies is compromised, but reinstalled by parallel depletion of EndoG. In sum, we unravel a phylogenetically conserved pathway that involves EndoG as a critical downstream executor of α-synuclein cytotoxicity.
Subject(s)
Apoptosis , Endodeoxyribonucleases/metabolism , Neuroblastoma/pathology , Neurons/metabolism , Parkinson Disease/pathology , Substantia Nigra/pathology , alpha-Synuclein/metabolism , Aged , Animals , Caenorhabditis elegans/growth & development , Caenorhabditis elegans/metabolism , DNA Damage/genetics , Dopamine/pharmacology , Drosophila melanogaster/growth & development , Drosophila melanogaster/metabolism , Endodeoxyribonucleases/genetics , Humans , Immunoblotting , Immunoenzyme Techniques , Mitochondria/metabolism , Mitochondria/pathology , Neuroblastoma/genetics , Neuroblastoma/metabolism , Neurons/cytology , Oxidative Stress , Parkinson Disease/genetics , Parkinson Disease/metabolism , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Substantia Nigra/metabolism , Tumor Cells, Cultured , alpha-Synuclein/geneticsABSTRACT
Mitochondria cannot form de novo but require mechanisms allowing their inheritance to daughter cells. In contrast to most other eukaryotes Trypanosoma brucei has a single mitochondrion whose single-unit genome is physically connected to the flagellum. Here we identify a ß-barrel mitochondrial outer membrane protein, termed tripartite attachment complex 40 (TAC40), that localizes to this connection. TAC40 is essential for mitochondrial DNA inheritance and belongs to the mitochondrial porin protein family. However, it is not specifically related to any of the three subclasses of mitochondrial porins represented by the metabolite transporter voltage-dependent anion channel (VDAC), the protein translocator of the outer membrane 40 (TOM40), or the fungi-specific MDM10, a component of the endoplasmic reticulum-mitochondria encounter structure (ERMES). MDM10 and TAC40 mediate cellular architecture and participate in transmembrane complexes that are essential for mitochondrial DNA inheritance. In yeast MDM10, in the context of the ERMES, is postulated to connect the mitochondrial genomes to actin filaments, whereas in trypanosomes TAC40 mediates the linkage of the mitochondrial DNA to the basal body of the flagellum. However, TAC40 does not colocalize with trypanosomal orthologs of ERMES components and, unlike MDM10, it regulates neither mitochondrial morphology nor the assembly of the protein translocase. TAC40 therefore defines a novel subclass of mitochondrial porins that is distinct from VDAC, TOM40, and MDM10. However, whereas the architecture of the TAC40-containing complex in trypanosomes and the MDM10-containing ERMES in yeast is very different, both are organized around a ß-barrel protein of the mitochondrial porin family that mediates a DNA-cytoskeleton linkage that is essential for mitochondrial DNA inheritance.
Subject(s)
Genes, Mitochondrial/genetics , Mitochondrial Membranes/metabolism , Mitochondrial Proteins/genetics , Models, Biological , Porins/genetics , Protozoan Proteins/genetics , Trypanosoma brucei brucei/genetics , Base Sequence , Cell Line , Cluster Analysis , Cytoskeleton/metabolism , DNA, Mitochondrial/metabolism , Fluorescent Antibody Technique , Mass Spectrometry , Microscopy, Electron, Transmission , Molecular Sequence Data , Organisms, Genetically Modified , Phylogeny , Sequence Analysis, DNA , Sequence HomologyABSTRACT
Mitochondrial outer membrane permeabilization is a watershed event in the process of apoptosis, which is tightly regulated by a series of pro- and anti-apoptotic proteins belonging to the BCL-2 family, each characteristically possessing a BCL-2 homology domain 3 (BH3). Here, we identify a yeast protein (Ybh3p) that interacts with BCL-X(L) and harbours a functional BH3 domain. Upon lethal insult, Ybh3p translocates to mitochondria and triggers BH3 domain-dependent apoptosis. Ybh3p induces cell death and disruption of the mitochondrial transmembrane potential via the mitochondrial phosphate carrier Mir1p. Deletion of Mir1p and depletion of its human orthologue (SLC25A3/PHC) abolish stress-induced mitochondrial targeting of Ybh3p in yeast and that of BAX in human cells, respectively. Yeast cells lacking YBH3 display prolonged chronological and replicative lifespans and resistance to apoptosis induction. Thus, the yeast genome encodes a functional BH3 domain that induces cell death through phylogenetically conserved mechanisms.
Subject(s)
Apoptosis Regulatory Proteins/metabolism , Apoptosis , Mitochondria/metabolism , Peptide Fragments/pharmacology , Proto-Oncogene Proteins/pharmacology , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Signal Transduction , Amino Acid Sequence , Animals , Apoptosis Regulatory Proteins/antagonists & inhibitors , Apoptosis Regulatory Proteins/genetics , Blotting, Western , Bone Neoplasms/genetics , Bone Neoplasms/metabolism , Cell Cycle , Flow Cytometry , Humans , Immunoprecipitation , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Membrane Potential, Mitochondrial , Mice , Mitochondria/genetics , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Molecular Sequence Data , Phosphate Transport Proteins/genetics , Phosphate Transport Proteins/metabolism , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Reverse Transcriptase Polymerase Chain Reaction , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/antagonists & inhibitors , Saccharomyces cerevisiae Proteins/genetics , Sequence Homology, Amino Acid , Tumor Cells, Cultured , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolism , bcl-X Protein/genetics , bcl-X Protein/metabolismABSTRACT
Trypanosoma brucei is a unicellular parasite that causes devastating diseases in humans and animals. It diverged from most other eukaryotes very early in evolution and, as a consequence, has an unusual mitochondrial biology. Moreover, mitochondrial functions and morphology are highly regulated throughout the life cycle of the parasite. The outer mitochondrial membrane defines the boundary of the organelle. Its properties are therefore key for understanding how the cytosol and mitochondria communicate and how the organelle is integrated into the metabolism of the whole cell. We have purified the mitochondrial outer membrane of T. brucei and characterized its proteome using label-free quantitative mass spectrometry for protein abundance profiling in combination with statistical analysis. Our results show that the trypanosomal outer membrane proteome consists of 82 proteins, two-thirds of which have never been associated with mitochondria before. 40 proteins share homology with proteins of known functions. The function of 42 proteins, 33 of which are specific to trypanosomatids, remains unknown. 11 proteins are essential for the disease-causing bloodstream form of T. brucei and therefore may be exploited as novel drug targets. A comparison with the outer membrane proteome of yeast defines a set of 17 common proteins that are likely present in the mitochondrial outer membrane of all eukaryotes. Known factors involved in the regulation of mitochondrial morphology are virtually absent in T. brucei. Interestingly, RNAi-mediated ablation of three outer membrane proteins of unknown function resulted in a collapse of the network-like mitochondrion of procyclic cells and for the first time identified factors that control mitochondrial shape in T. brucei.
Subject(s)
Mitochondria/metabolism , Mitochondria/ultrastructure , Mitochondrial Membranes/metabolism , Mitochondrial Proteins/genetics , Proteome/genetics , Protozoan Proteins/genetics , Trypanosoma brucei brucei/genetics , Animals , Gene Expression Profiling , Gene Expression Regulation , Humans , Life Cycle Stages/genetics , Mitochondria/genetics , Mitochondrial Membranes/chemistry , Mitochondrial Proteins/antagonists & inhibitors , Mitochondrial Proteins/metabolism , Organelle Shape/genetics , Proteome/antagonists & inhibitors , Proteome/metabolism , Protozoan Proteins/antagonists & inhibitors , Protozoan Proteins/metabolism , RNA, Small Interfering/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Tandem Mass Spectrometry , Trypanosoma brucei brucei/chemistry , Trypanosoma brucei brucei/metabolismABSTRACT
The intermembrane space (IMS) represents the smallest subcompartment of mitochondria. Nevertheless, it plays important roles in the transport and modification of proteins, lipids, and metal ions and in the regulation and assembly of the respiratory chain complexes. Moreover, it is involved in many redox processes and coordinates key steps in programmed cell death. A comprehensive profiling of IMS proteins has not been performed so far. We have established a method that uses the proapoptotic protein Bax to release IMS proteins from isolated mitochondria, and we profiled the protein composition of this compartment. Using stable isotope-labeled mitochondria from Saccharomyces cerevisiae, we were able to measure specific Bax-dependent protein release and distinguish between quantitatively released IMS proteins and the background efflux of matrix proteins. From the known 31 soluble IMS proteins, 29 proteins were reproducibly identified, corresponding to a coverage of >90%. In addition, we found 20 novel intermembrane space proteins, out of which 10 had not been localized to mitochondria before. Many of these novel IMS proteins have unknown functions or have been reported to play a role in redox regulation. We confirmed IMS localization for 15 proteins using in organello import, protease accessibility upon osmotic swelling, and Bax-release assays. Moreover, we identified two novel mitochondrial proteins, Ymr244c-a (Coa6) and Ybl107c (Mic23), as substrates of the MIA import pathway that have unusual cysteine motifs and found the protein phosphatase Ptc5 to be a novel substrate of the inner membrane protease (IMP). For Coa6 we discovered a role as a novel assembly factor of the cytochrome c oxidase complex. We present here the first and comprehensive proteome of IMS proteins of yeast mitochondria with 51 proteins in total. The IMS proteome will serve as a valuable source for further studies on the role of the IMS in cell life and death.
Subject(s)
Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Mitochondrial Proteins/analysis , Proteome/analysis , Saccharomyces cerevisiae Proteins/metabolism , Electron Transport Complex IV/metabolism , Gene Expression Profiling , Isotope Labeling , Protein Transport , Saccharomyces cerevisiae/physiology , bcl-2-Associated X Protein/metabolismABSTRACT
Mitochondria require an extensive proteome to maintain a variety of metabolic reactions, and changes in cellular demand depend on rapid adaptation of the mitochondrial protein composition. The TOM complex, the organellar entry gate for mitochondrial precursors in the outer membrane, is a target for cytosolic kinases to modulate protein influx. DYRK1A phosphorylation of the carrier import receptor TOM70 at Ser91 enables its efficient docking and thus transfer of precursor proteins to the TOM complex. Here, we probe TOM70 phosphorylation in molecular detail and find that TOM70 is not a CK2 target nor import receptor for MIC19 as previously suggested. Instead, we identify TOM20 as a MIC19 import receptor and show off-target inhibition of the DYRK1A-TOM70 axis with the clinically used CK2 inhibitor CX4945 which activates TOM20-dependent import pathways. Taken together, modulation of DYRK1A signalling adapts the central mitochondrial protein entry gate via synchronization of TOM70- and TOM20-dependent import pathways for metabolic rewiring. Thus, DYRK1A emerges as a cytosolic surveillance kinase to regulate and fine-tune mitochondrial protein biogenesis.