RESUMO
Mutations in PTEN-induced kinase 1 (PINK1) can cause recessive early-onset Parkinson's disease (PD). Import arrest results in PINK1 kinase activation specifically on damaged mitochondria, triggering Parkin-mediated mitophagy. Here, we show that PINK1 import is less dependent on Tim23 than on mitochondrial membrane potential (ΔΨm). We identified a negatively charged amino acid cluster motif that is evolutionarily conserved just C-terminal to the PINK1 transmembrane. PINK1 that fails to accumulate at the outer mitochondrial membrane, either by mutagenesis of this negatively charged motif or by deletion of Tom7, is imported into depolarized mitochondria and cleaved by the OMA1 protease. Some PD patient mutations also are defective in import arrest and are rescued by the suppression of OMA1, providing a new potential druggable target for PD. These results suggest that ΔΨm loss-dependent PINK1 import arrest does not result solely from Tim23 inactivation but also through an actively regulated "tug of war" between Tom7 and OMA1.
Assuntos
Proteínas de Membrana/metabolismo , Metaloendopeptidases/metabolismo , Mitocôndrias/enzimologia , Membranas Mitocondriais/enzimologia , Proteínas Mitocondriais/metabolismo , Doença de Parkinson/enzimologia , Proteínas Quinases/metabolismo , Motivos de Aminoácidos , Antiparkinsonianos/farmacologia , Transporte Biológico , Desenho de Fármacos , Ativação Enzimática , Células HeLa , Humanos , Potencial da Membrana Mitocondrial , Proteínas de Membrana/genética , Metaloendopeptidases/genética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/genética , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/genética , Domínios e Motivos de Interação entre Proteínas , Proteínas Quinases/genética , Proteólise , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Protein aggregates and damaged organelles are tagged with ubiquitin chains to trigger selective autophagy. To initiate mitophagy, the ubiquitin kinase PINK1 phosphorylates ubiquitin to activate the ubiquitin ligase parkin, which builds ubiquitin chains on mitochondrial outer membrane proteins, where they act to recruit autophagy receptors. Using genome editing to knockout five autophagy receptors in HeLa cells, here we show that two receptors previously linked to xenophagy, NDP52 and optineurin, are the primary receptors for PINK1- and parkin-mediated mitophagy. PINK1 recruits NDP52 and optineurin, but not p62, to mitochondria to activate mitophagy directly, independently of parkin. Once recruited to mitochondria, NDP52 and optineurin recruit the autophagy factors ULK1, DFCP1 and WIPI1 to focal spots proximal to mitochondria, revealing a function for these autophagy receptors upstream of LC3. This supports a new model in which PINK1-generated phospho-ubiquitin serves as the autophagy signal on mitochondria, and parkin then acts to amplify this signal. This work also suggests direct and broader roles for ubiquitin phosphorylation in other autophagy pathways.
Assuntos
Autofagia/fisiologia , Mitofagia/fisiologia , Proteínas Nucleares/metabolismo , Proteínas Quinases/metabolismo , Fator de Transcrição TFIIIA/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Mitochondrial dysfunction is associated with ageing, but the detailed causal relationship between the two is still unclear. We review the major phenomenological manifestations of mitochondrial age-related dysfunction including biochemical, regulatory and energetic features. We conclude that the complexity of these processes and their inter-relationships are still not fully understood and at this point it seems unlikely that a single linear cause and effect relationship between any specific aspect of mitochondrial biology and ageing can be established in either direction.
Assuntos
Senescência Celular , Mitocôndrias/metabolismo , Animais , Genoma Mitocondrial , Humanos , Mitocôndrias/genética , Biogênese de OrganelasRESUMO
Mitochondrial dysfunction is a causative and/or exacerbating feature of many pathologies. We discuss below approaches to modulate mitochondrial dysfunction that involve (1) increasing their energetic efficiency by targeting gene expression regulators such as PPAR or AMPK, (2) using antioxidant compounds to reduce the toxic reactive oxygen species mitochondria produce under stress, or (3) modulating aspects on the innate mitochondrial quality control system. The latter comprise linked processes of biogenesis, dynamic morphological changes, and elimination of defective mitochondria by mitophagy. We discuss representative compounds in all three classes.
Assuntos
Antioxidantes/farmacologia , Mitocôndrias/efeitos dos fármacos , Animais , Antioxidantes/química , Relação Dose-Resposta a Droga , Humanos , Mitocôndrias/metabolismo , Estrutura Molecular , Controle de Qualidade , Relação Estrutura-AtividadeRESUMO
Most of the mitochondrial inner-membrane proteins are generated without a presequence and their targeting depends on inadequately defined internal segments. Despite the numerous components of the import machinery identified by proteomics, the properties of hydrophobic import substrates remain poorly understood. Recent studies support several principles for these membrane proteins: first, they become organized into partially assembled forms within the translocon; second, they present noncontiguous targeting signals; and third, they induce conformational changes in translocase subunits, thereby mediating "assembly on demand" of the import machinery. It is possible that the energy needed for these proteins to pass across the outer membrane, to travel through the intermembrane space and to target the inner-membrane surface is provided by conformational changes involving import components that seem to have natively unfolded structures. Such structural malleability might render some of the translocase subunits more adept at driving the protein import process.
Assuntos
Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Conformação Molecular , Animais , Modelos Biológicos , Transporte ProteicoRESUMO
PINK1 and Parkin are established mediators of mitophagy, the selective removal of damaged mitochondria by autophagy. PINK1 and Parkin have been proposed to act as tumor suppressors, as loss-of-function mutations are correlated with enhanced tumorigenesis. However, it is unclear how PINK1 and Parkin act in coordination during mitophagy to influence the cell cycle. Here we show that PINK1 and Parkin genetically interact with proteins involved in cell cycle regulation, and loss of PINK1 and Parkin accelerates cell growth. PINK1- and Parkin-mediated activation of TBK1 at the mitochondria during mitophagy leads to a block in mitosis due to the sequestration of TBK1 from its physiological role at centrosomes during mitosis. Our study supports a diverse role for the far-reaching, regulatory effects of mitochondrial quality control in cellular homeostasis and demonstrates that the PINK1/Parkin pathway genetically interacts with the cell cycle, providing a framework for understanding the molecular basis linking PINK1 and Parkin to mitosis.
Assuntos
Ciclo Celular/genética , Mitocôndrias/genética , Mitose/genética , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Ubiquitina-Proteína Ligases/genética , Autofagia/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Células HCT116 , Células HEK293 , Células HeLa , Homeostase/genética , Humanos , Mitofagia/genéticaRESUMO
A new fluorescent Zn2+ indicator, namely, ICPBCZin was synthesized and the spectral profile of its free and Zn2+ bound forms was studied. The newly synthesized zinc indicator incorporates as chromophore the chromeno [3',2':3,4]pyrido[1,2a] [1,3]benzimidazole moiety and belongs to the dicarboxylate-type of zinc probes. The compound is excited with visible light, exhibits high selectivity for zinc in the presence of calcium and other common biological ions, and its Zn2+ dissociation constant is 4.0 nM. Fluorescence spectra studies of ICPBCZin indicated a clear shift in its emission wavelength maxima upon Zn2+ binding, as it belongs to the class of Photoinduced Charge Transfer (PCT) indicators, along with changes in fluorescence intensity that enable the compound to be used as a ratiometric, visible-excitable Zn2+ probe.
Assuntos
Benzimidazóis/química , Benzopiranos/química , Corantes Fluorescentes/química , Zinco/química , Benzimidazóis/síntese química , Benzopiranos/síntese química , Células Cultivadas , Quelantes/química , Corantes Fluorescentes/síntese química , Microscopia de Fluorescência , Espectrometria de FluorescênciaRESUMO
Unlike matrix-targeted or inner membrane proteins, those that are targeted to the mitochondrial intermembrane space (IMS) do not require ATP or the inner membrane electrochemical potential. Their import is mediated primarily by the essential IMS protein Mia40/Tim40. Here, we show that the mitochondrial flavin adenine dinucleotide (FAD)-linked sulfhydryl oxidase Erv1 (essential for respiration and vegetative growth 1) plays a central role in the biogenesis of small, cysteine proteins of the IMS that are import substrates for Mia40. In a temperature-sensitive strain of Erv1, steady-state levels of small translocases of the inner membrane (Tims) are specifically affected when cells are grown at the non-permissive temperature. Furthermore, mitochondria isolated from the erv1-ts show a specific import and assembly defect for the small Tims but not in any other protein import pathway. Erv1 does not directly oxidise the small Tims, as thiol trapping assays show that the small Tims can still be oxidised in erv1-ts cells grown at the non-permissive temperature and in isolated mitochondria from this strain. Moreover, addition of pure Erv1 into erv1-ts mitochondria lacking the endogenous protein restores import and assembly of the small Tims only to an extent, arguing for a cascade of interactions with Erv1 rather than for a direct interaction of Erv1 with the small Tims. Cytochrome c (cyt c) is the in vivo oxidase for Erv1, as yeast cells mutated in cyt c cannot grow under anaerobic conditions. Therefore, Erv1 functionally links the Mia40-dependent import pathway to the Mia40-independent cyt c import pathway transferring electrons from the incoming precursors to cyt c as an acceptor. In this context, the protein import process is linked to the respiratory chain via the communication of Erv1 with cyt c.
Assuntos
Citocromos c/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Transporte de Elétrons , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/química , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/fisiologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Transporte Proteico , TemperaturaRESUMO
Parkinson's disease (PD) is a neurodegenerative disease caused by the loss of dopaminergic neurons in the substantia nigra. PARK2 mutations cause early-onset forms of PD. PARK2 encodes an E3 ubiquitin ligase, Parkin, that can selectively translocate to dysfunctional mitochondria to promote their removal by autophagy. However, Parkin knockout (KO) mice do not display signs of neurodegeneration. To assess Parkin function in vivo, we utilized a mouse model that accumulates dysfunctional mitochondria caused by an accelerated generation of mtDNA mutations (Mutator mice). In the absence of Parkin, dopaminergic neurons in Mutator mice degenerated causing an L-DOPA reversible motor deficit. Other neuronal populations were unaffected. Phosphorylated ubiquitin was increased in the brains of Mutator mice, indicating PINK1-Parkin activation. Parkin loss caused mitochondrial dysfunction and affected the pathogenicity but not the levels of mtDNA somatic mutations. A systemic loss of Parkin synergizes with mitochondrial dysfunction causing dopaminergic neuron death modeling PD pathogenic processes.
Assuntos
DNA Mitocondrial/genética , Neurônios Dopaminérgicos/patologia , Doenças Mitocondriais/patologia , Mutação/genética , Substância Negra/patologia , Ubiquitina-Proteína Ligases/metabolismo , Análise de Variância , Animais , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Técnicas In Vitro , Levodopa/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Doenças Mitocondriais/genética , Complexos Multienzimáticos/metabolismo , Proteômica , Substância Negra/efeitos dos fármacos , Tirosina 3-Mono-Oxigenase/metabolismo , Ubiquitina , Ubiquitina-Proteína Ligases/genéticaRESUMO
The discovery of the mitochondrial intermembrane space assembly (MIA) pathway was followed by studies that focused mainly on the typical small substrates of this disulfide relay system and the interactions between its two central partners: the oxidoreductase Mia40 and the FAD-protein Erv1. Recent studies have revealed that more complex proteins utilize this pathway, including Mia40 itself. In the present study, we dissect the Mia40 biogenesis in distinct stages, supporting a kinetically coordinated sequence of events, starting with (a) import and insertion through the Tim23 translocon, followed by (b) folding of the core of imported Mia40 assisted by the endogenous Mia40 and (c) final interaction with Erv1. The interaction with endogenous Mia40 and the subsequent interaction with Erv1 represent kinetically distinguishable steps that rely on completely different determinants. Interaction with Mia40 proceeds very early (within 30 s) and is characterized by no Cys-specificity, an increased tolerance to mutations of the hydrophobic substrate-binding cleft and no apparent dependence on glutathione as a proofreading mechanism. All of these features illustrate a very atypical behaviour for the Mia40 precursor compared to other substrates of the MIA pathway. By contrast, interaction with Erv1 occurs after 5 min of import and relies on a more stringent specificity.
Assuntos
Proteínas de Transporte da Membrana Mitocondrial/biossíntese , Domínio Catalítico , Redutases do Citocromo/metabolismo , Glutationa/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Ligação Proteica , Dobramento de Proteína , Transporte ProteicoRESUMO
During autophagy, a double membrane envelops cellular material for trafficking to the lysosome. Human beclin-1 and its yeast homologue, Atg6/Vps30, are scaffold proteins bound in a lipid kinase complex with multiple cellular functions, including autophagy. Several different Atg6 complexes exist, with an autophagy-specific form containing Atg14. However, the roles of Atg14 and beclin-1 in the activation of this complex remain unclear. We here addressed the mechanism of beclin-1 complex activation and reveal two critical steps in this pathway. First, we identified a unique domain in beclin-1, conserved in the yeast homologue Atg6, which is involved in membrane association and, unexpectedly, controls autophagosome size and number in yeast. Second, we demonstrated that human Atg14 is critical in controlling an autophagy-dependent phosphorylation of beclin-1. We map these novel phosphorylation sites to serines 90 and 93 and demonstrate that phosphorylation at these sites is necessary for maximal autophagy. These results help clarify the mechanism of beclin-1 and Atg14 during autophagy.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/fisiologia , Proteínas de Membrana/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Adaptadoras de Transporte Vesicular/genética , Sequência de Aminoácidos , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/genética , Proteínas Relacionadas à Autofagia , Proteína Beclina-1 , Sítios de Ligação , Linhagem Celular , Técnicas de Inativação de Genes , Humanos , Membranas Intracelulares/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Dados de Sequência Molecular , Fagossomos/metabolismo , Fosforilação , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Serina/químicaRESUMO
The endoplasmic reticulum (ER) provides an environment optimized for oxidative protein folding through the action of Ero1p, which generates disulfide bonds, and Pdi1p, which receives disulfide bonds from Ero1p and transfers them to substrate proteins. Feedback regulation of Ero1p through reduction and oxidation of regulatory bonds within Ero1p is essential for maintaining the proper redox balance in the ER. In this paper, we show that Pdi1p is the key regulator of Ero1p activity. Reduced Pdi1p resulted in the activation of Ero1p by direct reduction of Ero1p regulatory bonds. Conversely, upon depletion of thiol substrates and accumulation of oxidized Pdi1p, Ero1p was inactivated by both autonomous oxidation and Pdi1p-mediated oxidation of Ero1p regulatory bonds. Pdi1p responded to the availability of free thiols and the relative levels of reduced and oxidized glutathione in the ER to control Ero1p activity and ensure that cells generate the minimum number of disulfide bonds needed for efficient oxidative protein folding.
Assuntos
Retículo Endoplasmático/metabolismo , Glicoproteínas/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Glutationa/metabolismo , Homeostase , Oxirredução , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The MIA40 pathway is a novel import pathway in mitochondria specific for cysteine-rich proteins of the intermembrane space (IMS). The newly synthesised precursors are trapped in the IMS by a disulfide relay mechanism that involves introduction of disulfides from the sulfhydryl oxidase Erv1 to the redox-regulated import receptor Mia40 and then on to the substrate. This thiol-disulfide exchange mechanism is essential for the import and oxidative folding of the incoming cysteine-rich substrate proteins. In this chapter we will describe the experimental methods that have been developed in order to study and characterise disulfide-trapped intermediates in yeast mitochondria.
Assuntos
Mitocôndrias/metabolismo , Proteínas/metabolismo , Autorradiografia , Eletroforese , Eletroforese em Gel de Poliacrilamida , Membranas Intracelulares/metabolismo , Modelos Biológicos , Oxirredução , Dobramento de Proteína , Transporte Proteico/fisiologia , Proteínas/química , Leveduras/metabolismoRESUMO
Disulfide bond formation is a crucial step for oxidative folding and necessary for the acquisition of a protein's native conformation. Introduction of disulfide bonds is catalyzed in specialized subcellular compartments and requires the coordinated action of specific enzymes. The intermembrane space of mitochondria has recently been found to harbor a dedicated machinery that promotes the oxidative folding of substrate proteins by shuttling disulfide bonds. The newly identified oxidative pathway consists of the redox-regulated receptor Mia40 and the sulfhydryl oxidase Erv1. Proteins destined to the intermembrane space are trapped by a disulfide relay mechanism that involves an electron cascade from the incoming substrate to Mia40, then on to Erv1, and finally to molecular oxygen via cytochrome c. This thiol-disulfide exchange mechanism is essential for the import and for maintaining the structural stability of the incoming precursors. In this review we describe the mechanistic parameters that define the interaction and oxidation of the substrate proteins in light of the recent publications in the mitochondrial oxidative folding field.
Assuntos
Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Dobramento de Proteína , Dissulfetos/química , Dissulfetos/metabolismo , Humanos , Proteínas de Transporte da Membrana Mitocondrial/química , Modelos Moleculares , OxirreduçãoRESUMO
Mia40 imports Cys-containing proteins into the mitochondrial intermembrane space (IMS) by ensuring their Cys-dependent oxidative folding. In this study, we show that the specific Cys of the substrate involved in docking with Mia40 is substrate dependent, the process being guided by an IMS-targeting signal (ITS) present in Mia40 substrates. The ITS is a 9-aa internal peptide that (a) is upstream or downstream of the docking Cys, (b) is sufficient for crossing the outer membrane and for targeting nonmitochondrial proteins, (c) forms an amphipathic helix with crucial hydrophobic residues on the side of the docking Cys and dispensable charged residues on the other side, and (d) fits complementary to the substrate cleft of Mia40 via hydrophobic interactions of micromolar affinity. We rationalize the dual function of Mia40 as a receptor and an oxidase in a two step-specific mechanism: an ITS-guided sliding step orients the substrate noncovalently, followed by docking of the substrate Cys now juxtaposed to pair with the Mia40 active Cys.
Assuntos
Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Dobramento de Proteína , Sinais Direcionadores de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Sítios de Ligação , Calorimetria , Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions , Sequência Consenso , Proteínas de Transporte de Cobre , Cisteína/química , Cisteína/metabolismo , Humanos , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Chaperonas Moleculares , Oxirredução , Estrutura Terciária de Proteína , Transporte Proteico , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/fisiologiaRESUMO
MIA40 has a key role in oxidative protein folding in the mitochondrial intermembrane space. We present the solution structure of human MIA40 and its mechanism as a catalyst of oxidative folding. MIA40 has a 66-residue folded domain made of an alpha-helical hairpin core stabilized by two structural disulfides and a rigid N-terminal lid, with a characteristic CPC motif that can donate its disulfide bond to substrates. The CPC active site is solvent-accessible and sits adjacent to a hydrophobic cleft. Its second cysteine (Cys55) is essential in vivo and is crucial for mixed disulfide formation with the substrate. The hydrophobic cleft functions as a substrate binding domain, and mutations of this domain are lethal in vivo and abrogate binding in vitro. MIA40 represents a thioredoxin-unrelated, minimal oxidoreductase, with a facile CPC redox active site that ensures its catalytic function in oxidative folding in mitochondria.
Assuntos
Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Sequência de Aminoácidos , Humanos , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Conformação Proteica , Dobramento de Proteína , Alinhamento de SequênciaRESUMO
Oxidative folding in the mitochondrial intermembrane space (IMS) is crucial for the import of certain cysteine-rich IMS proteins. The essential proteins Mia40 and Erv1 are key components for this mechanism functioning as a disulphide protein cascade that is functionally linked to the respiratory chain by shuttling electrons onto CytC. The subunits of the chaperone complex Tim9-Tim10 require Mia40 for their biogenesis. Previously, it was shown that the four cysteines of Tim10 are crucial for folding and assembly, that they are connected intramolecularly into an inner and an outer disulphide bridge, and that the inner disulphide has a more prominent role in these processes. Here we show that interaction with Mia40 is a site-specific event: (i) the N-terminal first cysteine of the precursor is crucial for docking onto Mia40 via a mixed disulphide; (ii) release is triggered by disulphide pairing of the C-terminal cysteine onto the N-terminal one; and (iii) formation of the inner disulphide between the second and third cysteines apparently precedes the release reaction and is critical for assembly with Tim9. The Tim10-Mia40 interaction is independent of divalent cations, any other mitochondrial proteins or membranes, and is shown to occur efficiently in organello and in vitro.