ABSTRACT
The mitochondrial F1FO-ATP synthase produces the bulk of cellular ATP. The soluble F1 domain contains the catalytic head that is linked via the central stalk and the peripheral stalk to the membrane embedded rotor of the FO domain. The assembly of the F1 domain and its linkage to the peripheral stalk is poorly understood. Here we show a dual function of the mitochondrial Hsp70 (mtHsp70) in the formation of the ATP synthase. First, it cooperates with the assembly factors Atp11 and Atp12 to form the F1 domain of the ATP synthase. Second, the chaperone transfers Atp5 into the assembly line to link the catalytic head with the peripheral stalk. Inactivation of mtHsp70 leads to integration of assembly-defective Atp5 variants into the mature complex, reflecting a quality control function of the chaperone. Thus, mtHsp70 acts as an assembly and quality control factor in the biogenesis of the F1FO-ATP synthase.
Subject(s)
Mitochondria , Mitochondrial Proton-Translocating ATPases , Mitochondrial Proton-Translocating ATPases/genetics , Mitochondrial Proton-Translocating ATPases/metabolism , Mitochondria/metabolism , Nitric Oxide Synthase , Adenosine TriphosphateABSTRACT
The presequence translocase (TIM23 complex) imports precursor proteins into the mitochondrial inner membrane and matrix. The presequence translocase-associated motor (PAM) provides a driving force for transport into the matrix. The J-protein Pam18 stimulates the ATPase activity of the mitochondrial Hsp70 (mtHsp70). Pam16 recruits Pam18 to the TIM23 complex to ensure protein import. The Pam16-Pam18 module also associates with components of the respiratory chain, but the function of the dual localization of Pam16-Pam18 is largely unknown. Here, we show that disruption of the Pam16-Pam18 heterodimer causes redistribution of Pam18 to the respiratory chain supercomplexes, where it forms a homodimer. Redistribution of Pam18 decreases protein import into mitochondria but stimulates mtHsp70-dependent assembly of respiratory chain complexes. We conclude that coupling to Pam16 differentially controls the dual function of Pam18. It recruits Pam18 to the TIM23 complex to promote protein import but attenuates the Pam18 function in the assembly of respiratory chain complexes.
Subject(s)
Membrane Transport Proteins , Mitochondrial Precursor Protein Import Complex Proteins , Saccharomyces cerevisiae Proteins , Carrier Proteins/metabolism , Electron Transport , HSP70 Heat-Shock Proteins/metabolism , Membrane Transport Proteins/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Precursor Protein Import Complex Proteins/metabolism , Protein Transport , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Substantial efforts have been undertaken to identify and minimise factors responsible for the development of ventilator-induced lung injury. A novel approach to this problem addresses energy dissipated in lung tissue during the breathing cycle as one of the key problems. Flow-controlled ventilation is a new modality of mechanical ventilation based on a constant flow during both inspiration and expiration. This review aims to evaluate the current evidence available regarding flow-controlled ventilation. Lastly, three cases of flow-controlled ventilation application are presented: ventilation with a small lumen tube during tracheal resection, one-lung ventilation during thoracoscopic lobectomy, and ventilation of a critically ill patient with acute respiratory distress syndrome in an intensive care unit setting.
Subject(s)
Respiratory Distress Syndrome , Ventilator-Induced Lung Injury , Humans , Lung , Respiration , Respiration, Artificial/methods , Respiratory Distress Syndrome/therapy , Ventilator-Induced Lung Injury/prevention & controlABSTRACT
Patient-derived human organoids can be used to model a variety of diseases. Recently, we described conditions for long-term expansion of human airway organoids (AOs) directly from healthy individuals and patients. Here, we first optimize differentiation of AOs towards ciliated cells. After differentiation of the AOs towards ciliated cells, these can be studied for weeks. When returned to expansion conditions, the organoids readily resume their growth. We apply this condition to AOs established from nasal inferior turbinate brush samples of patients suffering from primary ciliary dyskinesia (PCD), a pulmonary disease caused by dysfunction of the motile cilia in the airways. Patient-specific differences in ciliary beating are observed and are in agreement with the patients' genetic mutations. More detailed organoid ciliary phenotypes can thus be documented in addition to the standard diagnostic procedure. Additionally, using genetic editing tools, we show that a patient-specific mutation can be repaired. This study demonstrates the utility of organoid technology for investigating hereditary airway diseases such as PCD.
Subject(s)
Ciliary Motility Disorders , Organoids , Cilia , Ciliary Motility Disorders/genetics , Humans , Mutation , PhenotypeABSTRACT
Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes, where it is synthesized locally and plays an important role in mitochondrial bioenergetics. Previous studies in the yeast model have indicated that CL is required for optimal iron homeostasis, which is disrupted by a mechanism not yet determined in the yeast CL mutant, crd1Δ. This finding has implications for the severe genetic disorder, Barth syndrome (BTHS), in which CL metabolism is perturbed because of mutations in the CL-remodeling enzyme, tafazzin. Here, we investigate the effects of tafazzin deficiency on iron homeostasis in the mouse myoblast model of BTHS tafazzin knockout (TAZ-KO) cells. Similarly to CL-deficient yeast cells, TAZ-KO cells exhibited elevated sensitivity to iron, as well as to H2O2, which was alleviated by the iron chelator deferoxamine. TAZ-KO cells exhibited increased expression of the iron exporter ferroportin and decreased expression of the iron importer transferrin receptor, likely reflecting a regulatory response to elevated mitochondrial iron. Reduced activities of mitochondrial iron-sulfur cluster enzymes suggested that the mechanism underlying perturbation of iron homeostasis was defective iron-sulfur biogenesis. We observed decreased levels of Yfh1/frataxin, an essential component of the iron-sulfur biogenesis machinery, in mitochondria from TAZ-KO mouse cells and in CL-deleted yeast crd1Δ cells, indicating that the role of CL in iron-sulfur biogenesis is highly conserved. Yeast crd1Δ cells exhibited decreased processing of the Yfh1 precursor upon import, which likely contributes to the iron homeostasis defects. Implications for understanding the pathogenesis of BTHS are discussed.
Subject(s)
Barth Syndrome/metabolism , Cardiolipins/metabolism , Iron-Binding Proteins/metabolism , Iron/metabolism , Myoblasts/metabolism , Acyltransferases , Animals , Barth Syndrome/genetics , Barth Syndrome/pathology , Cardiolipins/genetics , Cell Line , Gene Deletion , Gene Knockout Techniques , Iron-Binding Proteins/genetics , Mice , Myoblasts/pathology , Transcription Factors/genetics , Transcription Factors/metabolism , FrataxinABSTRACT
Organoids are self-organizing 3D structures grown from stem cells that recapitulate essential aspects of organ structure and function. Here, we describe a method to establish long-term-expanding human airway organoids from broncho-alveolar resections or lavage material. The pseudostratified airway organoids consist of basal cells, functional multi-ciliated cells, mucus-producing secretory cells, and CC10-secreting club cells. Airway organoids derived from cystic fibrosis (CF) patients allow assessment of CFTR function in an organoid swelling assay. Organoids established from lung cancer resections and metastasis biopsies retain tumor histopathology as well as cancer gene mutations and are amenable to drug screening. Respiratory syncytial virus (RSV) infection recapitulates central disease features, dramatically increases organoid cell motility via the non-structural viral NS2 protein, and preferentially recruits neutrophils upon co-culturing. We conclude that human airway organoids represent versatile models for the in vitro study of hereditary, malignant, and infectious pulmonary disease.
Subject(s)
Carcinoma, Non-Small-Cell Lung/pathology , Cystic Fibrosis/pathology , Epithelial Cells/pathology , Organ Culture Techniques/methods , Organoids/pathology , Respiratory Syncytial Virus Infections/pathology , Respiratory System/pathology , Animals , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/metabolism , Cells, Cultured , Cystic Fibrosis/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Disease Models, Animal , Drug Screening Assays, Antitumor , Epithelial Cells/metabolism , Female , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Male , Mice , Mice, Inbred NOD , Mice, SCID , Organoids/metabolism , Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Viruses/isolation & purification , Respiratory System/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Mitochondria are the powerhouses of eukaryotic cells. The activity of the respiratory chain complexes generates a proton gradient across the inner membrane, which is used by the F1FO-ATP synthase to produce ATP for cellular metabolism. In baker's yeast, Saccharomyces cerevisiae, the cytochrome bc1 complex (complex III) and cytochrome c oxidase (complex IV) associate in respiratory chain supercomplexes. Iron-sulfur clusters (ISC) form reactive centers of respiratory chain complexes. The assembly of ISC occurs in the mitochondrial matrix and is essential for cell viability. The cysteine desulfurase Nfs1 provides sulfur for ISC assembly and forms with partner proteins the ISC-biogenesis desulfurase complex (ISD complex). Here, we report an unexpected interaction of the active ISD complex with the cytochrome bc1 complex and cytochrome c oxidase. The individual deletion of complex III or complex IV blocks the association of the ISD complex with respiratory chain components. We conclude that the ISD complex binds selectively to respiratory chain supercomplexes. We propose that this molecular link contributes to coordination of iron-sulfur cluster formation with respiratory activity.
ABSTRACT
A functional crosstalk between epigenetic regulators and metabolic control could provide a mechanism to adapt cellular responses to environmental cues. We report that the well-known nuclear MYST family acetyl transferase MOF and a subset of its non-specific lethal complex partners reside in mitochondria. MOF regulates oxidative phosphorylation by controlling expression of respiratory genes from both nuclear and mtDNA in aerobically respiring cells. MOF binds mtDNA, and this binding is dependent on KANSL3. The mitochondrial pool of MOF, but not a catalytically deficient mutant, rescues respiratory and mtDNA transcriptional defects triggered by the absence of MOF. Mof conditional knockout has catastrophic consequences for tissues with high-energy consumption, triggering hypertrophic cardiomyopathy and cardiac failure in murine hearts; cardiomyocytes show severe mitochondrial degeneration and deregulation of mitochondrial nutrient metabolism and oxidative phosphorylation pathways. Thus, MOF is a dual-transcriptional regulator of nuclear and mitochondrial genomes connecting epigenetics and metabolism.
Subject(s)
Energy Metabolism/genetics , Epigenesis, Genetic , Histone Acetyltransferases/metabolism , Mitochondria, Muscle/enzymology , Transcription Factors/metabolism , Transcription, Genetic , Animals , Cardiomyopathy, Hypertrophic/genetics , Cell Respiration/genetics , DNA, Mitochondrial/genetics , DNA, Mitochondrial/metabolism , HeLa Cells , Heart Failure/genetics , Histone Acetyltransferases/genetics , Humans , Intracellular Signaling Peptides and Proteins , Mice , Mice, Knockout , Mitochondria, Heart/enzymology , Mitochondria, Heart/genetics , Mitochondria, Muscle/genetics , Myocytes, Cardiac/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Oxidative Phosphorylation , Transcription Factors/geneticsABSTRACT
Mitochondria have to import the vast majority of their proteins, which are synthesized as precursors on cytosolic ribosomes. The translocase of the outer membrane (TOM complex) forms the general entry gate for the precursor proteins, which are subsequently sorted by protein machineries into the mitochondrial subcompartments: the outer and inner membrane, the intermembrane space and the mitochondrial matrix. The transport across and into the inner membrane is driven by the membrane potential, which is generated by the respiratory chain. Recent studies revealed that the lipid composition of mitochondrial membranes is important for the biogenesis of mitochondrial proteins. Cardiolipin and phosphatidylethanolamine exhibit unexpectedly specific functions for the activity of distinct protein translocases. Both phospholipids are required for full activity of respiratory chain complexes and thus to maintain the membrane potential for protein import. In addition, cardiolipin is required to maintain structural integrity of mitochondrial protein translocases. Finally, the low sterol content in the mitochondrial outer membrane may contribute to the targeting of some outer membrane proteins with a single α-helical membrane anchor. Altogether, mitochondrial lipids modulate protein import on various levels involving precursor targeting, membrane potential generation, stability and activity of protein translocases.
Subject(s)
Lipids , Membrane Potential, Mitochondrial/physiology , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Mitochondrial Proteins/metabolism , Animals , Humans , Protein Transport/physiologyABSTRACT
Two protein translocases drive the import of ß-barrel precursor proteins into the mitochondrial outer membrane: The translocase of the outer membrane (TOM complex) promotes transport of the precursor to the intermembrane space, whereas the sorting and assembly machinery (SAM complex) mediates subsequent folding of the ß-barrel and its integration into the target membrane. The non-bilayer-forming phospholipids phosphatidylethanolamine (PE) and cardiolipin (CL) are required for the biogenesis of ß-barrel proteins. Whether bilayer-forming phospholipids such as phosphatidylcholine (PC), the most abundant phospholipid of the mitochondrial outer membrane, play a role in the import of ß-barrel precursors is unclear. In this study, we show that PC is required for stability and function of the SAM complex during the biogenesis of ß-barrel proteins. PC further promotes the SAM-dependent assembly of the TOM complex, indicating a general role of PC for the function of the SAM complex. In contrast to PE-deficient mitochondria precursor accumulation at the TOM complex is not affected by depletion of PC. We conclude that PC and PE affect the function of distinct protein translocases in mitochondrial ß-barrel biogenesis.
Subject(s)
Mitochondrial Proteins/metabolism , Phosphatidylcholines/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Mitochondrial Proteins/genetics , Phosphatidylcholines/genetics , Protein Transport/physiology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
The content of mitochondrial proteome is maintained through two highly dynamic processes, the influx of newly synthesized proteins from the cytosol and the protein degradation. Mitochondrial proteins are targeted to the intermembrane space by the mitochondrial intermembrane space assembly pathway that couples their import and oxidative folding. The folding trap was proposed to be a driving mechanism for the mitochondrial accumulation of these proteins. Whether the reverse movement of unfolded proteins to the cytosol occurs across the intact outer membrane is unknown. We found that reduced, conformationally destabilized proteins are released from mitochondria in a size-limited manner. We identified the general import pore protein Tom40 as an escape gate. We propose that the mitochondrial proteome is not only regulated by the import and degradation of proteins but also by their retro-translocation to the external cytosolic location. Thus, protein release is a mechanism that contributes to the mitochondrial proteome surveillance.
Subject(s)
Mitochondrial Membranes/metabolism , Mitochondrial Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Mitochondrial Proteins/chemistry , Oxidation-Reduction , Protein Conformation , Protein Folding , Protein Transport , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistryABSTRACT
Mitochondrial Hsp70 (mtHsp70) mediates essential functions for mitochondrial biogenesis, like import and folding of proteins. In these processes, the chaperone cooperates with cochaperones, the presequence translocase, and other chaperone systems. The chaperonin Hsp60, together with its cofactor Hsp10, catalyzes folding of a subset of mtHsp70 client proteins. Hsp60 forms heptameric ring structures that provide a cavity for protein folding. How the Hsp60 rings are assembled is poorly understood. In a comprehensive interaction study, we found that mtHsp70 associates with Hsp60 and Hsp10. Surprisingly, mtHsp70 interacts with Hsp10 independently of Hsp60. The mtHsp70-Hsp10 complex binds to the unassembled Hsp60 precursor to promote its assembly into mature Hsp60 complexes. We conclude that coupling to Hsp10 recruits mtHsp70 to mediate the biogenesis of the heptameric Hsp60 rings.
Subject(s)
Chaperonin 10/metabolism , Chaperonin 60/metabolism , HSP70 Heat-Shock Proteins/metabolism , Mitochondria/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Animals , Chaperonin 60/chemistry , Saccharomyces cerevisiae/cytologyABSTRACT
The mitochondrial outer membrane contains integral α-helical and ß-barrel proteins that are imported from the cytosol. The machineries importing ß-barrel proteins have been identified, however, different views exist on the import of α-helical proteins. It has been reported that the biogenesis of Om45, the most abundant signal-anchored protein, does not depend on proteinaceous components, but involves direct insertion into the outer membrane. We show that import of Om45 occurs via the translocase of the outer membrane and the presequence translocase of the inner membrane. Assembly of Om45 in the outer membrane involves the MIM machinery. Om45 thus follows a new mitochondrial biogenesis pathway that uses elements of the presequence import pathway to direct a protein to the outer membrane.
Subject(s)
Carrier Proteins/metabolism , Membrane Proteins/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Membranes/physiology , Protein Transport/physiology , Saccharomyces cerevisiae Proteins/metabolism , Autoradiography , Electrophoresis, Polyacrylamide Gel , Membrane Proteins/chemistry , Membrane Transport Proteins/metabolism , Mitochondrial Precursor Protein Import Complex Proteins , Mutagenesis , Polymerase Chain Reaction , Protein Structure, Secondary , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins/chemistryABSTRACT
The formation of the mature cytochrome c oxidase (complex IV) involves the association of nuclear- and mitochondria-encoded subunits. The assembly of nuclear-encoded subunits like cytochrome c oxidase subunit 4 (Cox4) into the mature complex is poorly understood. Cox4 is crucial for the stability of complex IV. To find specific biogenesis factors, we analyze interaction partners of Cox4 by affinity purification and mass spectroscopy. Surprisingly, we identify a complex of Cox4, the mitochondrial Hsp70 (mtHsp70), and its nucleotide-exchange factor mitochondrial GrpE (Mge1). We generate a yeast mutant of mtHsp70 specifically impaired in the formation of this novel mtHsp70-Mge1-Cox4 complex. Strikingly, the assembly of Cox4 is strongly decreased in these mutant mitochondria. Because Cox4 is a key factor for the biogenesis of complex IV, we conclude that the mtHsp70-Mge1-Cox4 complex plays an important role in the formation of cytochrome c oxidase. Cox4 arrests at this chaperone complex in the absence of mature complex IV. Thus the mtHsp70-Cox4 complex likely serves as a novel delivery system to channel Cox4 into the assembly line when needed.
Subject(s)
Electron Transport Complex IV/metabolism , HSP70 Heat-Shock Proteins/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Molecular Chaperones/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Electron Transport Complex IV/chemistry , HSP70 Heat-Shock Proteins/genetics , Membrane Proteins/metabolism , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins/chemistry , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/metabolism , Molecular Chaperones/chemistry , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
The mitochondrial outer membrane contains proteinaceous machineries for the import and assembly of proteins, including TOM (translocase of the outer membrane) and SAM (sorting and assembly machinery). It has been shown that the dimeric phospholipid cardiolipin is required for the stability of TOM and SAM complexes and thus for the efficient import and assembly of ß-barrel proteins and some α-helical proteins of the outer membrane. Here, we report that mitochondria deficient in phosphatidylethanolamine (PE), the second non-bilayer-forming phospholipid, are impaired in the biogenesis of ß-barrel proteins, but not of α-helical outer membrane proteins. The stability of TOM and SAM complexes is not disturbed by the lack of PE. By dissecting the import steps of ß-barrel proteins, we show that an early import stage involving translocation through the TOM complex is affected. In PE-depleted mitochondria, the TOM complex binds precursor proteins with reduced efficiency. We conclude that PE is required for the proper function of the TOM complex.
Subject(s)
Carrier Proteins/metabolism , Membrane Proteins/metabolism , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Phosphatidylethanolamines/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Carrier Proteins/genetics , Membrane Proteins/genetics , Mitochondria/genetics , Mitochondrial Precursor Protein Import Complex Proteins , Mitochondrial Proteins/genetics , Phosphatidylethanolamines/genetics , Protein Structure, Secondary , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
The inner mitochondrial membrane plays a crucial role in cellular lipid homeostasis through biosynthesis of the non-bilayer-forming lipids phosphatidylethanolamine and cardiolipin. In the yeast Saccharomyces cerevisiae, the majority of cellular phosphatidylethanolamine is synthesized by the mitochondrial phosphatidylserine decarboxylase 1 (Psd1). The biogenesis of Psd1 involves several processing steps. It was speculated that the Psd1 precursor is sorted into the inner membrane and is subsequently released into the intermembrane space by proteolytic removal of a hydrophobic sorting signal. However, components involved in the maturation of the Psd1 precursor have not been identified. We show that processing of Psd1 involves the action of the mitochondrial processing peptidase and Oct1 and an autocatalytic cleavage at a highly conserved LGST motif yielding the α- and ß-subunit of the enzyme. The Psd1 ß-subunit (Psd1ß) forms the membrane anchor, which binds the intermembrane space-localized α-subunit (Psd1α). Deletion of a transmembrane segment in the ß-subunit results in mislocalization of Psd1 and reduced enzymatic activity. Surprisingly, autocatalytic cleavage does not depend on proper localization to the inner mitochondrial membrane. In summary, membrane integration of Psd1 is crucial for its functionality and for maintenance of mitochondrial lipid homeostasis.
Subject(s)
Carboxy-Lyases/metabolism , Mitochondrial Membranes/enzymology , Mitochondrial Proteins/metabolism , Protein Processing, Post-Translational , Protein Subunits/metabolism , Saccharomyces cerevisiae/enzymology , Amino Acid Sequence , Carboxy-Lyases/chemistry , Membrane Potential, Mitochondrial , Mitochondria/enzymology , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Precursor Protein Import Complex Proteins , Mitochondrial Proteins/chemistry , Organic Cation Transporter 1/metabolism , Protein Precursors/chemistry , Protein Precursors/metabolism , Protein Sorting Signals , Protein Structure, Tertiary , Protein Subunits/chemistry , Protein Transport , Proteolysis , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
The mitochondrial inner membrane contains two non-bilayer-forming phospholipids, phosphatidylethanolamine (PE) and cardiolipin (CL). Lack of CL leads to destabilization of respiratory chain supercomplexes, a reduced activity of cytochrome c oxidase, and a reduced inner membrane potential Δψ. Although PE is more abundant than CL in the mitochondrial inner membrane, its role in biogenesis and assembly of inner membrane complexes is unknown. We report that similar to the lack of CL, PE depletion resulted in a decrease of Δψ and thus in an impaired import of preproteins into and across the inner membrane. The respiratory capacity and in particular the activity of cytochrome c oxidase were impaired in PE-depleted mitochondria, leading to the decrease of Δψ. In contrast to depletion of CL, depletion of PE did not destabilize respiratory chain supercomplexes but favored the formation of larger supercomplexes (megacomplexes) between the cytochrome bc(1) complex and the cytochrome c oxidase. We conclude that both PE and CL are required for a full activity of the mitochondrial respiratory chain and the efficient generation of the inner membrane potential. The mechanisms, however, are different since these non-bilayer-forming phospholipids exert opposite effects on the stability of respiratory chain supercomplexes.
Subject(s)
Cardiolipins/physiology , Electron Transport/physiology , Mitochondria/physiology , Phosphatidylethanolamines/physiology , Electron Transport Complex IV/metabolism , Mitochondria/enzymology , Protein TransportABSTRACT
The intermembrane space of mitochondria accommodates the essential mitochondrial intermembrane space assembly (MIA) machinery that catalyzes oxidative folding of proteins. The disulfide bond formation pathway is based on a relay of reactions involving disulfide transfer from the sulfhydryl oxidase Erv1 to Mia40 and from Mia40 to substrate proteins. However, the substrates of the MIA typically contain two disulfide bonds. It was unclear what the mechanisms are that ensure that proteins are released from Mia40 in a fully oxidized form. In this work, we dissect the stage of the oxidative folding relay, in which Mia40 binds to its substrate. We identify dynamics of the Mia40-substrate intermediate complex. Our experiments performed in a native environment, both in organello and in vivo, show that Erv1 directly participates in Mia40-substrate complex dynamics by forming a ternary complex. Thus Mia40 in cooperation with Erv1 promotes the formation of two disulfide bonds in the substrate protein, ensuring the efficiency of oxidative folding in the intermembrane space of mitochondria.