ABSTRACT
The exchange of metabolites between the mitochondrial matrix and the cytosol depends on ß-barrel channels in the outer membrane and α-helical carrier proteins in the inner membrane. The essential translocase of the inner membrane (TIM) chaperones escort these proteins through the intermembrane space, but the structural and mechanistic details remain elusive. We have used an integrated structural biology approach to reveal the functional principle of TIM chaperones. Multiple clamp-like binding sites hold the mitochondrial membrane proteins in a translocation-competent elongated form, thus mimicking characteristics of co-translational membrane insertion. The bound preprotein undergoes conformational dynamics within the chaperone binding clefts, pointing to a multitude of dynamic local binding events. Mutations in these binding sites cause cell death or growth defects associated with impairment of carrier and ß-barrel protein biogenesis. Our work reveals how a single mitochondrial "transfer-chaperone" system is able to guide α-helical and ß-barrel membrane proteins in a "nascent chain-like" conformation through a ribosome-free compartment.
Subject(s)
Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Molecular Chaperones/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Amino Acid Sequence , Binding Sites , Intracellular Membranes/metabolism , Mitochondrial Membrane Transport Proteins/chemistry , Mitochondrial Membrane Transport Proteins/genetics , Molecular Dynamics Simulation , Mutagenesis, Site-Directed , Protein Binding , Protein Domains , Protein Precursors/chemistry , Protein Precursors/metabolism , Protein Structure, Secondary , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Sequence AlignmentABSTRACT
The respiratory chain in aerobic organisms is composed of a number of membrane-bound protein complexes that link electron transfer to proton translocation across the membrane. In mitochondria, the final electron acceptor, complex IV (CIV), receives electrons from dimeric complex III (CIII2), via a mobile electron carrier, cytochrome c. In the present study, we isolated the CIII2CIV supercomplex from the fission yeast Schizosaccharomyces pombe and determined its structure with bound cyt. c using single-particle electron cryomicroscopy. A respiratory supercomplex factor 2 was found to be bound at CIV distally positioned in the supercomplex. In addition to the redox-active metal sites, we found a metal ion, presumably Zn2+, coordinated in the CIII subunit Cor1, which is encoded by the same gene (qcr1) as the mitochondrial-processing peptidase subunit ß. Our data show that the isolated CIII2CIV supercomplex displays proteolytic activity suggesting a dual role of CIII2 in S. pombe. As in the supercomplex from S. cerevisiae, subunit Cox5 of CIV faces towards one CIII monomer, but in S. pombe, the two complexes are rotated relative to each other by ~45°. This orientation yields equal distances between the cyt. c binding sites at CIV and at each of the two CIII monomers. The structure shows cyt. c bound at four positions, but only along one of the two symmetrical branches. Overall, this combined structural and functional study reveals the integration of peptidase activity with the CIII2 respiratory system and indicates a two-dimensional cyt. c diffusion mechanism within the CIII2-CIV supercomplex.
Subject(s)
Schizosaccharomyces , Schizosaccharomyces/metabolism , Saccharomyces cerevisiae/metabolism , Cytochromes c/metabolism , Mitochondria/metabolism , Electron Transport Complex IV/metabolism , Electron Transport , Peptide Hydrolases/metabolism , Electron Transport Complex III/metabolismABSTRACT
While targeting of proteins synthesized in the cytosol to any organelle is complex, mitochondria present the most challenging of destinations. First, import of nuclear-encoded proteins needs to be balanced with production of mitochondrial-encoded ones. Moreover, as mitochondria are divided into distinct subdomains, their proteins harbor a number of different targeting signals and biophysical properties. While translocation into the mitochondrial membranes has been well studied, the cytosolic steps of protein import remain poorly understood. Here, we review current knowledge on mRNA and protein targeting to mitochondria, as well as recent advances in our understanding of the cellular programs that respond to accumulation of mitochondrial precursor proteins in the cytosol, thus linking defects in targeting-capacity to signaling.
Subject(s)
Cytosol/metabolism , Mitochondrial Proteins/biosynthesis , Heat-Shock Proteins/metabolism , Homeostasis , Mitochondrial Proteins/metabolism , Protein Biosynthesis , Protein Processing, Post-Translational , Protein Transport , Signal Recognition Particle/metabolism , Signal TransductionABSTRACT
Approximately half of eukaryotic proteins reside in organelles. To reach their correct destination, such proteins harbor targeting signals recognized by dedicated targeting pathways. It has been shown that differences in targeting signals alter the efficiency in which proteins are recognized and targeted. Since multiple proteins compete for any single pathway, such differences can affect the priority for which a protein is catered. However, to date the entire repertoire of proteins with targeting priority, and the mechanisms underlying it, have not been explored for any pathway. Here we developed a systematic tool to study targeting priority and used the Pex5-mediated targeting to yeast peroxisomes as a model. We titrated Pex5 out by expressing high levels of a Pex5-cargo protein and examined how the localization of each peroxisomal protein is affected. We found that while most known Pex5 cargo proteins were outcompeted, several cargo proteins were not affected, implying that they have high targeting priority. This priority group was dependent on metabolic conditions. We dissected the mechanism of priority for these proteins and suggest that targeting priority is governed by different parameters, including binding affinity of the targeting signal to the cargo factor, the number of binding interfaces to the cargo factor, and more. This approach can be modified to study targeting priority in various organelles, cell types, and organisms.
Subject(s)
Peroxisomal Targeting Signals , Peroxisome-Targeting Signal 1 Receptor/metabolism , Peroxisomes/metabolism , Proof of Concept Study , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
The version of Supplementary Table 1 originally published online with this article contained incorrect localization annotations for one plate. This error has been corrected in the online Supplementary Information.
ABSTRACT
Mitochondria originated from proteobacterial endosymbionts, and their transition to organelles was tightly linked to establishment of the protein import pathways. The initial import of most proteins is mediated by the translocase of the outer membrane (TOM). Although TOM is common to all forms of mitochondria, an unexpected diversity of subunits between eukaryotic lineages has been predicted. However, experimental knowledge is limited to a few organisms, and so far, it remains unsettled whether the triplet-pore or the twin-pore structure is the generic form of TOM complex. Here, we analysed the TOM complex in hydrogenosomes, a metabolically specialised anaerobic form of mitochondria found in the excavate Trichomonas vaginalis. We demonstrate that the highly divergent ß-barrel T. vaginalis TOM (TvTom)40-2 forms a translocation channel to conduct hydrogenosomal protein import. TvTom40-2 is present in high molecular weight complexes, and their analysis revealed the presence of four tail-anchored (TA) proteins. Two of them, Tom36 and Tom46, with heat shock protein (Hsp)20 and tetratricopeptide repeat (TPR) domains, can bind hydrogenosomal preproteins and most likely function as receptors. A third subunit, Tom22-like protein, has a short cis domain and a conserved Tom22 transmembrane segment but lacks a trans domain. The fourth protein, hydrogenosomal outer membrane protein 19 (Homp19) has no known homology. Furthermore, our data indicate that TvTOM is associated with sorting and assembly machinery (Sam)50 that is involved in ß-barrel assembly. Visualisation of TvTOM by electron microscopy revealed that it forms three pores and has an unconventional skull-like shape. Although TvTOM seems to lack Tom7, our phylogenetic profiling predicted Tom7 in free-living excavates. Collectively, our results suggest that the triplet-pore TOM complex, composed of three conserved subunits, was present in the last common eukaryotic ancestor (LECA), while receptors responsible for substrate binding evolved independently in different eukaryotic lineages.
Subject(s)
Carrier Proteins/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Trichomonas vaginalis/metabolism , Carrier Proteins/genetics , Carrier Proteins/physiology , Membrane Proteins/metabolism , Membrane Transport Proteins/metabolism , Mitochondria/metabolism , Mitochondrial Precursor Protein Import Complex Proteins , Organelles , Phylogeny , Protein Transport/physiology , Trichomonas vaginalis/pathogenicity , Trichomonas vaginalis/physiologyABSTRACT
Yeast libraries revolutionized the systematic study of cell biology. To extensively increase the number of such libraries, we used our previously devised SWAp-Tag (SWAT) approach to construct a genome-wide library of ~5,500 strains carrying the SWAT NOP1promoter-GFP module at the N terminus of proteins. In addition, we created six diverse libraries that restored the native regulation, created an overexpression library with a Cherry tag, or enabled protein complementation assays from two fragments of an enzyme or fluorophore. We developed methods utilizing these SWAT collections to systematically characterize the yeast proteome for protein abundance, localization, topology, and interactions.
Subject(s)
Genome, Fungal , Genomic Library , Proteome/genetics , Saccharomyces cerevisiae/genetics , Genetic Complementation Test , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Promoter Regions, Genetic , Protein Interaction Mapping , Proteome/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Ribonucleoproteins, Small Nucleolar/genetics , Ribonucleoproteins, Small Nucleolar/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Sequence Tagged SitesABSTRACT
Tail-anchored (TA) proteins are embedded into their corresponding membrane via a single transmembrane segment at their C-terminus whereas the majority of the protein is facing the cytosol. So far, cellular factors that mediate the integration of such proteins into the mitochondrial outer membrane were not found. Using budding yeast as a model system, we identified the cytosolic Hsp70 chaperone Ssa1 and the peroxisome import factor Pex19 as import mediators for a subset of mitochondrial TA proteins. Accordingly, deletion of PEX19 results in: (1) growth defect under respiration conditions, (2) alteration in mitochondrial morphology, (3) reduced steady-state levels of the mitochondrial TA proteins Fis1 and Gem1, and (4) hampered in organello import of the TA proteins Fis1 and Gem1. Furthermore, recombinant Pex19 can bind directly the TA proteins Fis1 and Gem1. Collectively, this work identified the first factors that are involved in the biogenesis of mitochondrial TA proteins and uncovered an unexpected function of Pex19.
Subject(s)
Membrane Proteins/metabolism , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Peroxisomes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , HSP70 Heat-Shock Proteins/metabolism , Heat-Shock Proteins/metabolism , Mitochondria/ultrastructure , Peroxisomes/ultrastructure , Protein Binding , Protein Transport , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/ultrastructureABSTRACT
Tail-anchored (TA) proteins are anchored to their corresponding membrane via a single transmembrane segment (TMS) at their C-terminus. In yeast, the targeting of TA proteins to the endoplasmic reticulum (ER) can be mediated by the guided entry of TA proteins (GET) pathway, whereas it is not yet clear how mitochondrial TA proteins are targeted to their destination. It has been widely observed that some mitochondrial outer membrane (MOM) proteins are mistargeted to the ER when overexpressed or when their targeting signal is masked. However, the mechanism of this erroneous sorting is currently unknown. In this study, we demonstrate the involvement of the GET machinery in the mistargeting of suboptimal MOM proteins to the ER. These findings suggest that the GET machinery can, in principle, recognize and guide mitochondrial and non-canonical TA proteins. Hence, under normal conditions, an active mitochondrial targeting pathway must exist that dominates the kinetic competition against other pathways.
Subject(s)
Adaptor Proteins, Vesicular Transport/metabolism , Adenosine Triphosphatases/metabolism , Endoplasmic Reticulum/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Membrane Proteins/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Adaptor Proteins, Vesicular Transport/genetics , Endoplasmic Reticulum/genetics , Membrane Proteins/genetics , Mitochondrial Membrane Transport Proteins/genetics , Mitochondrial Membranes/metabolism , Protein Transport , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Mitochondria harbor in their outer membrane (OM) proteins of different topologies. These proteins are encoded by the nuclear DNA, translated on cytosolic ribosomes and inserted into their target organelle by sophisticated protein import machineries. Recently, considerable insights have been accumulated on the insertion pathways of proteins into the mitochondrial OM. In contrast, little is known regarding the early cytosolic stages of their biogenesis. It is generally presumed that chaperones associate with these proteins following their synthesis in the cytosol, thereby keeping them in an import-competent conformation and preventing their aggregation and/or mis-folding and degradation. In this review, we outline the current knowledge about the biogenesis of different mitochondrial OM proteins with various topologies, and highlight the recent findings regarding their import pathways starting from early cytosolic events until their recognition on the mitochondrial surface that lead to their final insertion into the mitochondrial OM.
Subject(s)
Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Animals , Humans , Mitochondrial Proteins/chemistry , Protein Conformation, alpha-Helical , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/metabolismABSTRACT
In Gram-negative bacteria, secretion of toxins ensure the survival of the bacterium. Such toxins are secreted by sophisticated multiprotein systems. The most conserved part in some of these secretion systems are components, called secretins, which form the outer membrane ring in these systems. Recent structural studies shed some light on the oligomeric organization of secretins. However, the mechanisms by which these proteins are targeted to the outer membrane and assemble there into ring structures are still not fully understood. This review discusses the various species-specific targeting and assembly pathways that are taken by secretins in order to form their functional oligomers.
Subject(s)
Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/metabolism , Bacterial Outer Membrane/metabolism , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/metabolism , Bacterial Outer Membrane/chemistry , Bacterial Secretion Systems/chemistry , Bacterial Secretion Systems/metabolism , Gram-Negative Bacteria/chemistry , Gram-Negative Bacteria/metabolism , Models, Biological , Models, Molecular , Molecular Chaperones/chemistry , Molecular Chaperones/metabolism , Protein Multimerization , Protein TransportABSTRACT
The protease HtrA2 has a protective role inside mitochondria, but promotes apoptosis under stress. We previously identified the G399S HtrA2 mutation in Parkinson's disease (PD) patients and reported mitochondrial dysfunction in vitro. Mitochondrial dysfunction is a common feature of PD and related to neurodegeneration. Complete loss of HtrA2 has been shown to cause neurodegeneration in mice. However, the full impact of HtrA2 overexpression or the G399S mutation is still to be determined in vivo. Here, we report the first HtrA2 G399S transgenic mouse model. Our data suggest that the mutation has a dominant-negative effect. We also describe a toxic effect of wild-type (WT) HtrA2 overexpression. Only low overexpression of the G399S mutation allowed viable animals and we suggest that the mutant protein is likely unstable. This is accompanied by reduced mitochondrial respiratory capacity and sensitivity to apoptotic cell death. Mice overexpressing WT HtrA2 were viable, yet these animals have inhibited mitochondrial respiration and significant induction of apoptosis in the brain leading to motor dysfunction, highlighting the opposing roles of HtrA2. Our data further underscore the importance of HtrA2 as a key mediator of mitochondrial function and its fine regulatory role in cell fate. The location and abundance of HtrA2 is tightly controlled and, therefore, human mutations leading to gain- or loss of function could provide significant risk for PD-related neurodegeneration.
Subject(s)
Electron Transport Chain Complex Proteins/genetics , Mitochondria/genetics , Mitochondrial Proteins/genetics , Mutation , Parkinson Disease/genetics , Serine Endopeptidases/genetics , Animals , Apoptosis , Brain/metabolism , Brain/pathology , Cell Respiration , Disease Models, Animal , Electron Transport Chain Complex Proteins/metabolism , Female , Gene Dosage , Gene Expression Regulation , High-Temperature Requirement A Serine Peptidase 2 , Humans , Male , Mice , Mice, Transgenic , Mitochondria/metabolism , Mitochondria/pathology , Mitochondrial Proteins/metabolism , Motor Activity , Neurons/metabolism , Neurons/pathology , Parkinson Disease/metabolism , Parkinson Disease/pathology , Phenotype , Serine Endopeptidases/metabolismABSTRACT
Mitochondria are separated from the remainder of the eukaryotic cell by the mitochondrial outer membrane (MOM). The MOM plays an important role in different transport processes like lipid trafficking and protein import. In yeast, the ER-mitochondria encounter structure (ERMES) has a central, but poorly defined role in both activities. To understand the functions of the ERMES, we searched for suppressors of the deficiency of one of its components, Mdm10, and identified a novel mitochondrial protein that we named Mdm10 complementing protein 3 (Mcp3). Mcp3 partially rescues a variety of ERMES-related phenotypes. We further demonstrate that Mcp3 is an integral protein of the MOM that follows a unique import pathway. It is recognized initially by the import receptor Tom70 and then crosses the MOM via the translocase of the outer membrane. Mcp3 is next relayed to the TIM23 translocase at the inner membrane, gets processed by the inner membrane peptidase (IMP) and finally integrates into the MOM. Hence, Mcp3 follows a novel biogenesis route where a MOM protein is processed by a peptidase of the inner membrane.
Subject(s)
Fungal Proteins/metabolism , Mitochondrial Membranes/metabolism , Mitochondrial Proteins/metabolism , Peptide Hydrolases/metabolism , Signal Transduction , Carrier Proteins/metabolism , Fungal Proteins/genetics , Gene Deletion , Gene Dosage , Gene Expression , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mitochondria/genetics , Mitochondria/metabolism , Mitochondria/ultrastructure , Mitochondrial Precursor Protein Import Complex Proteins , Mitochondrial Proteins/genetics , Multiprotein Complexes , Protein Transport , ProteolysisABSTRACT
Diabetes mellitus and neurodegeneration are common diseases for which shared genetic factors are still only partly known. Here, we show that loss of the BiP (immunoglobulin heavy-chain binding protein) co-chaperone DNAJC3 leads to diabetes mellitus and widespread neurodegeneration. We investigated three siblings with juvenile-onset diabetes and central and peripheral neurodegeneration, including ataxia, upper-motor-neuron damage, peripheral neuropathy, hearing loss, and cerebral atrophy. Exome sequencing identified a homozygous stop mutation in DNAJC3. Screening of a diabetes database with 226,194 individuals yielded eight phenotypically similar individuals and one family carrying a homozygous DNAJC3 deletion. DNAJC3 was absent in fibroblasts from all affected subjects in both families. To delineate the phenotypic and mutational spectrum and the genetic variability of DNAJC3, we analyzed 8,603 exomes, including 506 from families affected by diabetes, ataxia, upper-motor-neuron damage, peripheral neuropathy, or hearing loss. This analysis revealed only one further loss-of-function allele in DNAJC3 and no further associations in subjects with only a subset of the features of the main phenotype. Our findings demonstrate that loss-of-function DNAJC3 mutations lead to a monogenic, recessive form of diabetes mellitus in humans. Moreover, they present a common denominator for diabetes and widespread neurodegeneration. This complements findings from mice in which knockout of Dnajc3 leads to diabetes and modifies disease in a neurodegenerative model of Marinesco-Sjögren syndrome.
Subject(s)
Diabetes Mellitus, Type 1/genetics , Gene Expression Regulation , HSP40 Heat-Shock Proteins/genetics , Heat-Shock Proteins/genetics , Multiple System Atrophy/genetics , Adolescent , Adult , Ataxia/genetics , Diabetes Mellitus, Type 1/diagnostic imaging , Endoplasmic Reticulum Chaperone BiP , Exome/genetics , Female , Fibroblasts , HSP40 Heat-Shock Proteins/metabolism , Homozygote , Humans , Male , Models, Molecular , Multiple System Atrophy/diagnostic imaging , Mutation , Pedigree , Phenotype , Radiography , Sequence Analysis, DNA , Young AdultABSTRACT
Mitochondria are unique organelles that contain their own - although strongly reduced - genome, and are surrounded by two membranes. While most cellular phospholipid biosynthesis takes place in the ER, mitochondria harbor the whole spectrum of glycerophospholipids common to biological membranes. Mitochondria also contribute to overall phospholipid biosynthesis in cells by producing phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. Considering these features, it is not surprising that mitochondria maintain highly active exchange of phospholipids with other cellular compartments. In this contribution we describe the transport of phospholipids between mitochondria and other organelles, and discuss recent developments in our understanding of the molecular functions of the protein complexes that mediate these processes. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.
Subject(s)
Mitochondria/metabolism , Phospholipids/metabolism , Animals , Biological Transport/physiology , Cardiolipins/metabolism , Endoplasmic Reticulum/metabolism , Humans , Membrane Proteins/metabolism , Mitochondrial Membranes/metabolism , Phosphatidylethanolamines/metabolismABSTRACT
Regulation of the localization of mRNAs and local translation are universal features in eukaryotes and contribute to cellular asymmetry and differentiation. In Saccharomyces cerevisiae, localization of mRNAs that encode membrane proteins requires the She protein machinery, including the RNA-binding protein She2p, as well as movement of the cortical endoplasmic reticulum (cER) to the yeast bud. In a screen for ER-specific proteins necessary for the directional transport of WSC2 and EAR1 mRNAs, we have identified enzymes that are involved in phospholipid metabolism. Loss of the phospholipid methyltransferase Cho2p, which showed the strongest impact on mRNA localization, disturbs mRNA localization, as well as ER morphology and segregation, owing to an increase in the amount of cellular phosphatidylethanolamine (PtdEtn). Mislocalized mRNPs containing She2p colocalize with aggregated cER structures, suggestive of the entrapment of mRNA and She2p by the elevated PtdEtn level. This was confirmed by the elevated binding of She2p to PtdEtn-containing liposomes. These findings underscore the importance of ER membrane integrity in mRNA transport.
Subject(s)
Endoplasmic Reticulum/metabolism , Phosphatidylethanolamine N-Methyltransferase/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/physiology , Lipid Metabolism/genetics , Liposomes/metabolism , Phosphatidylethanolamine N-Methyltransferase/genetics , Phosphatidylethanolamines/metabolism , Protein Binding , Protein Transport , RNA Transport , RNA, Messenger/genetics , RNA-Binding Proteins/genetics , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Yersinia adhesin A (YadA) belongs to a class of bacterial adhesins that form trimeric structures. Their mature form contains a passenger domain and a C-terminal ß-domain that anchors the protein in the outer membrane (OM). Little is known about how precursors of such proteins cross the periplasm and assemble into the OM. In the present study we took advantage of the evolutionary conservation in the biogenesis of ß-barrel proteins between bacteria and mitochondria. We previously observed that upon expression in yeast cells, bacterial ß-barrel proteins including the transmembrane domain of YadA assemble into the mitochondrial OM. In the current study we found that when expressed in yeast cells both the monomeric and trimeric forms of full-length YadA were detected in mitochondria but only the trimeric species was fully integrated into the OM. The oligomeric form was exposed on the surface of the organelle in its native conformation and maintained its capacity to adhere to host cells. The co-expression of YadA with a mitochondria-targeted form of the bacterial periplasmic chaperone Skp, but not with SurA or SecB, resulted in enhanced levels of both forms of YadA. Taken together, these results indicate that the proper assembly of trimeric autotransporter can occur also in a system lacking the lipoproteins of the BAM machinery and is specifically enhanced by the chaperone Skp.
Subject(s)
Adhesins, Bacterial/metabolism , Conserved Sequence , Evolution, Molecular , Mitochondria/metabolism , Protein Multimerization , Adhesins, Bacterial/chemistry , HeLa Cells , Humans , Mitochondrial Membranes/metabolism , Mitochondrial Proteins/metabolism , Molecular Chaperones/metabolism , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Transport , Proteolysis , Saccharomyces cerevisiae/metabolismABSTRACT
The yeast mitochondrial outer membrane (MOM) protein Mdm10 is involved in at least three different processes: (1) association of mitochondria with the endoplasmic reticulum and mitochondrial lipid homeostasis (2) membrane assembly of MOM proteins, and (3) inheritance and morphogenesis of mitochondria. To decipher the precise role of Mdm10 in mitochondrial function, we screened for high-copy suppressors of the severe growth defect of the mdm10Δ mutant. We identified two novel mitochondrial proteins (open reading frames YOR228c and YLR253w) that we named Mdm10 complementing protein (Mcp) 1 and Mcp2. Overexpression of Mcp1 or Mcp2 restores the alterations in morphology and stability of respiratory chain complexes of mitochondria devoid of Mdm10, but the observed defect in assembly of MOM proteins is not rescued. Lipid analysis demonstrates that elevated levels of Mcp1 and Mcp2 restore the alterations in mitochondrial phospholipid and ergosterol homeostasis in cells lacking Mdm10. Collectively, this work identifies two novel proteins that play a role in mitochondrial lipid homeostasis and describes a role of Mdm10 in ergosterol trafficking.
Subject(s)
Lipid Metabolism , Macrophages/metabolism , Microtubule-Associated Proteins/metabolism , Mitochondrial Membranes/metabolism , Mitochondrial Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Homeostasis , Saccharomyces cerevisiae/metabolismABSTRACT
The vast majority of outer membrane (OM) proteins in Gram-negative bacteria belongs to the class of membrane-embedded ß-barrel proteins. Besides Gram-negative bacteria, the presence of ß-barrel proteins is restricted to the OM of the eukaryotic organelles mitochondria and chloroplasts that were derived from prokaryotic ancestors. The assembly of these proteins into the corresponding OM is in each case facilitated by a dedicated protein complex that contains a highly conserved central ß-barrel protein termed BamA/YaeT/Omp85 in Gram-negative bacteria and Tob55/Sam50 in mitochondria. However, little is known about the exact mechanism by which these complexes mediate the integration of ß-barrel precursors into the lipid bilayer. Interestingly, previous studies showed that during evolution, these complexes retained the ability to functionally assemble ß-barrel proteins from different origins. In this review we summarize the current knowledge on the biogenesis pathway of ß-barrel proteins in Gram-negative bacteria, mitochondria and chloroplasts and focus on the commonalities and divergences that evolved between the different ß-barrel assembly machineries.
Subject(s)
Bacterial Outer Membrane Proteins/metabolism , Chloroplast Proteins/metabolism , Mitochondrial Proteins/metabolism , Protein Multimerization , Bacterial Outer Membrane Proteins/chemistry , Biological Evolution , Chloroplast Proteins/chemistry , Chloroplasts/chemistry , Chloroplasts/metabolism , Gram-Negative Bacteria/chemistry , Gram-Negative Bacteria/metabolism , Mitochondria/chemistry , Mitochondria/metabolism , Mitochondrial Membranes/chemistry , Mitochondrial Membranes/metabolism , Mitochondrial Proteins/chemistry , Models, Biological , Protein ConformationABSTRACT
Deafness-Dystonia-Optic Neuropathy (DDON) Syndrome is a rare X-linked progressive neurodegenerative disorder resulting from mutations in the TIMM8A gene encoding for the deafness dystonia protein 1 (DDP1). Despite important progress in identifying and characterizing novel mutations in this gene, little is known about the underlying pathomechanisms. Deficiencies in the biogenesis of hTim23 and consecutive alterations in biogenesis of inner membrane and matrix proteins have been proposed to serve as one possible mechanistic explanation. To shed new light on the role of DDP1 in the biogenesis of mammalian mitochondria, we investigated the effects of reduced or elevated DDP1 levels on mitochondrial dynamics and function. Our results show a reduction in the import of ß-barrel proteins into mitochondria from cells overexpressing DDP1. This effect was not observed when the DDON-related mutant form DDP1-C66W was overexpressed. Live cell microscopy of primary fibroblasts derived from DDON patients and of DDP1 downregulated HeLa cells displayed alterations of mitochondrial morphology with notable extensions in the length of mitochondrial tubules, whereas overexpression of DDP1 induced the formation of hollow spherical mitochondria. Of note, knockdown of the TIMM8A gene by RNA interference did not show an influence on the oxygen respiration rate and the mitochondrial membrane potential. Taken together, these results suggest that alterations in the levels of DDP1 can affect the morphology of mitochondria and thus shed new light on the pathogenic mechanisms of DDON.