RESUMO
Distinct morphologies of the mitochondrial network support divergent metabolic and regulatory processes that determine cell function and fate1-3. The mechanochemical GTPase optic atrophy 1 (OPA1) influences the architecture of cristae and catalyses the fusion of the mitochondrial inner membrane4,5. Despite its fundamental importance, the molecular mechanisms by which OPA1 modulates mitochondrial morphology are unclear. Here, using a combination of cellular and structural analyses, we illuminate the molecular mechanisms that are key to OPA1-dependent membrane remodelling and fusion. Human OPA1 embeds itself into cardiolipin-containing membranes through a lipid-binding paddle domain. A conserved loop within the paddle domain inserts deeply into the bilayer, further stabilizing the interactions with cardiolipin-enriched membranes. OPA1 dimerization through the paddle domain promotes the helical assembly of a flexible OPA1 lattice on the membrane, which drives mitochondrial fusion in cells. Moreover, the membrane-bending OPA1 oligomer undergoes conformational changes that pull the membrane-inserting loop out of the outer leaflet and contribute to the mechanics of membrane remodelling. Our findings provide a structural framework for understanding how human OPA1 shapes mitochondrial morphology and show us how human disease mutations compromise OPA1 functions.
Assuntos
GTP Fosfo-Hidrolases , Fusão de Membrana , Mitocôndrias , Membranas Mitocondriais , Humanos , Biocatálise , Cardiolipinas/química , Cardiolipinas/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Mitocôndrias/química , Mitocôndrias/metabolismo , Membranas Mitocondriais/química , Membranas Mitocondriais/enzimologia , Membranas Mitocondriais/metabolismo , Mutação , Domínios Proteicos , Multimerização Proteica , Dinâmica MitocondrialRESUMO
The presequence translocase of the mitochondrial inner membrane (TIM23) represents the major route for the import of nuclear-encoded proteins into mitochondria1,2. About 60% of more than 1,000 different mitochondrial proteins are synthesized with amino-terminal targeting signals, termed presequences, which form positively charged amphiphilic α-helices3,4. TIM23 sorts the presequence proteins into the inner membrane or matrix. Various views, including regulatory and coupling functions, have been reported on the essential TIM23 subunit Tim17 (refs. 5-7). Here we mapped the interaction of Tim17 with matrix-targeted and inner membrane-sorted preproteins during translocation in the native membrane environment. We show that Tim17 contains conserved negative charges close to the intermembrane space side of the bilayer, which are essential to initiate presequence protein translocation along a distinct transmembrane cavity of Tim17 for both classes of preproteins. The amphiphilic character of mitochondrial presequences directly matches this Tim17-dependent translocation mechanism. This mechanism permits direct lateral release of transmembrane segments of inner membrane-sorted precursors into the inner membrane.
Assuntos
Mitocôndrias , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial/metabolismo , Transporte Proteico , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The mitochondrial outer membrane harbors two protein translocases that are essential for cell viability: the translocase of the outer mitochondrial membrane (TOM) and the sorting and assembly machinery (SAM). The precursors of ß-barrel proteins use both translocases-TOM for import to the intermembrane space and SAM for export into the outer membrane. It is unknown if the translocases cooperate and where the ß-barrel of newly imported proteins is formed. We established a position-specific assay for monitoring ß-barrel formation in vivo and in organello and demonstrated that the ß-barrel was formed and membrane inserted while the precursor was bound to SAM. ß-barrel formation was inhibited by SAM mutants and, unexpectedly, by mutants of the central import receptor, Tom22. We show that the cytosolic domain of Tom22 links TOM and SAM into a supercomplex, facilitating precursor transfer on the intermembrane space side. Our study reveals receptor-mediated coupling of import and export translocases as a means of precursor channeling.
Assuntos
Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo , Transporte Proteico , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas Mitocondriais/química , Mutação , Porinas/química , Porinas/metabolismo , Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
In this issue of Molecular Cell, Roh et al. (2018) present a high-resolution cryo-EM structure of the nanodisc-reconstituted yeast Vo proton channel that provides important new insights into subunit arrangements and the proton translocation pathway in V-type ATPases.
Assuntos
Prótons , ATPases Vacuolares Próton-Translocadoras , Microscopia Crioeletrônica , Transporte de Íons , Saccharomyces cerevisiaeRESUMO
Balanced fusion and fission are key for the proper function and physiology of mitochondria1,2. Remodelling of the mitochondrial inner membrane is mediated by the dynamin-like protein mitochondrial genome maintenance 1 (Mgm1) in fungi or the related protein optic atrophy 1 (OPA1) in animals3-5. Mgm1 is required for the preservation of mitochondrial DNA in yeast6, whereas mutations in the OPA1 gene in humans are a common cause of autosomal dominant optic atrophy-a genetic disorder that affects the optic nerve7,8. Mgm1 and OPA1 are present in mitochondria as a membrane-integral long form and a short form that is soluble in the intermembrane space. Yeast strains that express temperature-sensitive mutants of Mgm19,10 or mammalian cells that lack OPA1 display fragmented mitochondria11,12, which suggests that Mgm1 and OPA1 have an important role in inner-membrane fusion. Consistently, only the mitochondrial outer membrane-not the inner membrane-fuses in the absence of functional Mgm113. Mgm1 and OPA1 have also been shown to maintain proper cristae architecture10,14; for example, OPA1 prevents the release of pro-apoptotic factors by tightening crista junctions15. Finally, the short form of OPA1 localizes to mitochondrial constriction sites, where it presumably promotes mitochondrial fission16. How Mgm1 and OPA1 perform their diverse functions in membrane fusion, scission and cristae organization is at present unknown. Here we present crystal and electron cryo-tomography structures of Mgm1 from Chaetomium thermophilum. Mgm1 consists of a GTPase (G) domain, a bundle signalling element domain, a stalk, and a paddle domain that contains a membrane-binding site. Biochemical and cell-based experiments demonstrate that the Mgm1 stalk mediates the assembly of bent tetramers into helical filaments. Electron cryo-tomography studies of Mgm1-decorated lipid tubes and fluorescence microscopy experiments on reconstituted membrane tubes indicate how the tetramers assemble on positively or negatively curved membranes. Our findings convey how Mgm1 and OPA1 filaments dynamically remodel the mitochondrial inner membrane.
Assuntos
Chaetomium/química , Microscopia Crioeletrônica , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas de Ligação ao GTP/química , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/química , Cristalografia por Raios X , Proteínas Fúngicas/ultraestrutura , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/ultraestrutura , Galactosilceramidas/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/ultraestrutura , Modelos Moleculares , Domínios Proteicos , Multimerização ProteicaRESUMO
Death receptor-mediated apoptosis requires the mitochondrial apoptosis pathway in many mammalian cells. In response to death receptor signaling, the truncated BH3-only protein BID can activate the proapoptotic BCL-2 proteins BAX and BAK and trigger the permeabilization of the mitochondria. BAX and BAK are inhibited by prosurvival BCL-2 proteins through retrotranslocation from the mitochondria into the cytosol, but a specific resistance mechanism to truncated BID-dependent apoptosis is unknown. Here, we report that hexokinase 1 and hexokinase 2 inhibit the apoptosis activator truncated BID as well as the effectors BAX and BAK by retrotranslocation from the mitochondria into the cytosol. BCL-2 protein shuttling and protection from TRAIL- and FasL-induced cell death requires mitochondrial hexokinase localization and interactions with the BH3 motifs of BCL-2 proteins but not glucose phosphorylation. Together, our work establishes hexokinase-dependent retrotranslocation of truncated BID as a selective protective mechanism against death receptor-induced apoptosis on the mitochondria.
Assuntos
Apoptose/fisiologia , Hexoquinase/metabolismo , Mitocôndrias/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Antibacterianos/farmacologia , Antibióticos Antineoplásicos/farmacologia , Linhagem Celular , Ciclosporina/farmacologia , Dactinomicina/farmacologia , Doxorrubicina/farmacologia , Inibidores Enzimáticos/farmacologia , Proteína Ligante Fas/farmacologia , Deleção de Genes , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hexoquinase/genética , Humanos , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/genéticaRESUMO
BACKGROUND: Barth syndrome (BTHS) is caused by mutations of the gene encoding tafazzin, which catalyzes maturation of mitochondrial cardiolipin and often manifests with systolic dysfunction during early infancy. Beyond the first months of life, BTHS cardiomyopathy typically transitions to a phenotype of diastolic dysfunction with preserved ejection fraction, blunted contractile reserve during exercise, and arrhythmic vulnerability. Previous studies traced BTHS cardiomyopathy to mitochondrial formation of reactive oxygen species (ROS). Because mitochondrial function and ROS formation are regulated by excitation-contraction coupling, integrated analysis of mechano-energetic coupling is required to delineate the pathomechanisms of BTHS cardiomyopathy. METHODS: We analyzed cardiac function and structure in a mouse model with global knockdown of tafazzin (Taz-KD) compared with wild-type littermates. Respiratory chain assembly and function, ROS emission, and Ca2+ uptake were determined in isolated mitochondria. Excitation-contraction coupling was integrated with mitochondrial redox state, ROS, and Ca2+ uptake in isolated, unloaded or preloaded cardiac myocytes, and cardiac hemodynamics analyzed in vivo. RESULTS: Taz-KD mice develop heart failure with preserved ejection fraction (>50%) and age-dependent progression of diastolic dysfunction in the absence of fibrosis. Increased myofilament Ca2+ affinity and slowed cross-bridge cycling caused diastolic dysfunction, in part, compensated by accelerated diastolic Ca2+ decay through preactivated sarcoplasmic reticulum Ca2+-ATPase. Taz deficiency provoked heart-specific loss of mitochondrial Ca2+ uniporter protein that prevented Ca2+-induced activation of the Krebs cycle during ß-adrenergic stimulation, oxidizing pyridine nucleotides and triggering arrhythmias in cardiac myocytes. In vivo, Taz-KD mice displayed prolonged QRS duration as a substrate for arrhythmias, and a lack of inotropic response to ß-adrenergic stimulation. Cellular arrhythmias and QRS prolongation, but not the defective inotropic reserve, were restored by inhibiting Ca2+ export through the mitochondrial Na+/Ca2+ exchanger. All alterations occurred in the absence of excess mitochondrial ROS in vitro or in vivo. CONCLUSIONS: Downregulation of mitochondrial Ca2+ uniporter, increased myofilament Ca2+ affinity, and preactivated sarcoplasmic reticulum Ca2+-ATPase provoke mechano-energetic uncoupling that explains diastolic dysfunction and the lack of inotropic reserve in BTHS cardiomyopathy. Furthermore, defective mitochondrial Ca2+ uptake provides a trigger and a substrate for ventricular arrhythmias. These insights can guide the ongoing search for a cure of this orphaned disease.
Assuntos
Arritmias Cardíacas/diagnóstico , Arritmias Cardíacas/etiologia , Síndrome de Barth/complicações , Síndrome de Barth/genética , Canais de Cálcio/deficiência , Contração Miocárdica/genética , Trifosfato de Adenosina/biossíntese , Animais , Síndrome de Barth/metabolismo , Biomarcadores , Encéfalo/metabolismo , Cálcio/metabolismo , Diástole , Modelos Animais de Doenças , Suscetibilidade a Doenças , Acoplamento Excitação-Contração/genética , Testes de Função Cardíaca , Humanos , Camundongos , Camundongos Knockout , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/metabolismo , Músculo Esquelético/metabolismo , Miócitos Cardíacos/metabolismo , NADP/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Volume Sistólico , SístoleRESUMO
The majority of preproteins destined for mitochondria carry N-terminal presequences. The presequence translocase of the inner mitochondrial membrane (TIM23 complex) plays a central role in protein sorting. Preproteins are either translocated through the TIM23 complex into the matrix or are laterally released into the inner membrane. We report that the small hydrophobic protein Mgr2 controls the lateral release of preproteins. Mgr2 interacts with preproteins in transit through the TIM23 complex. Overexpression of Mgr2 delays preprotein release, whereas a lack of Mgr2 promotes preprotein sorting into the inner membrane. Preproteins with a defective inner membrane sorting signal are translocated into the matrix in wild-type mitochondria but are released into the inner membrane in Mgr2-deficient mitochondria. We conclude that Mgr2 functions as a lateral gatekeeper of the mitochondrial presequence translocase, providing quality control for the membrane sorting of preproteins.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Membrana/genética , Metotrexato/farmacologia , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/metabolismo , Transporte Proteico/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
BACKGROUND: The mitochondrial pyruvate carrier (MPC) plays a central role in energy metabolism by transporting pyruvate across the inner mitochondrial membrane. Its heterodimeric composition and homology to SWEET and semiSWEET transporters set the MPC apart from the canonical mitochondrial carrier family (named MCF or SLC25). The import of the canonical carriers is mediated by the carrier translocase of the inner membrane (TIM22) pathway and is dependent on their structure, which features an even number of transmembrane segments and both termini in the intermembrane space. The import pathway of MPC proteins has not been elucidated. The odd number of transmembrane segments and positioning of the N-terminus in the matrix argues against an import via the TIM22 carrier pathway but favors an import via the flexible presequence pathway. RESULTS: Here, we systematically analyzed the import pathways of Mpc2 and Mpc3 and report that, contrary to an expected import via the flexible presequence pathway, yeast MPC proteins with an odd number of transmembrane segments and matrix-exposed N-terminus are imported by the carrier pathway, using the receptor Tom70, small TIM chaperones, and the TIM22 complex. The TIM9·10 complex chaperones MPC proteins through the mitochondrial intermembrane space using conserved hydrophobic motifs that are also required for the interaction with canonical carrier proteins. CONCLUSIONS: The carrier pathway can import paired and non-paired transmembrane helices and translocate N-termini to either side of the mitochondrial inner membrane, revealing an unexpected versatility of the mitochondrial import pathway for non-cleavable inner membrane proteins.
Assuntos
Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte BiológicoRESUMO
Mitochondria are complex double-membrane-bound organelles of eukaryotic cells that function as energy-converting powerhouses, metabolic factories and signaling centers. The outer membrane controls the exchange of material and information with other cellular compartments. The inner membrane provides an extended, highly folded surface for selective transport and energy-coupling reactions. It can be divided into an inner boundary membrane and tubular or lamellar cristae membranes that accommodate the oxidative phosphorylation units. Outer membrane, inner boundary membrane and cristae come together at crista junctions, where the mitochondrial contact site and cristae organizing system (MICOS) acts as a membrane-shaping and -connecting scaffold. This peculiar architecture is of pivotal importance for multiple mitochondrial functions. Many elaborate studies in the past years have shed light on the subunit composition and organization of MICOS. In this review article, we summarize these insights and then move on to discuss exciting recent discoveries on the integrative functions of MICOS. Multi-faceted connections to other major players of mitochondrial biogenesis and physiology, like the protein import machineries, the oxidative phosphorylation system, carrier proteins and phospholipid biosynthesis enzymes, are currently emerging. Therefore, we propose that MICOS acts as a central hub in mitochondrial membrane architecture and functionality.
Assuntos
Mitocôndrias/genética , Humanos , Mitocôndrias/metabolismo , Transdução de SinaisRESUMO
Metabolic remodeling is a major determinant for many cell fate decisions, and a switch from respiration to aerobic glycolysis is generally considered as a hallmark of cancer cell transformation. Pyruvate is a key metabolite at the major junction of carbohydrate metabolism between cytosolic glycolysis and the mitochondrial Krebs cycle. In this issue of The EMBO Journal, Bender et al show that yeast cells regulate pyruvate uptake into mitochondria, and thus its metabolic fate, by expressing alternative pyruvate carrier complexes with different activities.
Assuntos
Proteínas de Transporte de Ânions/biossíntese , Proteínas de Membrana/biossíntese , Mitocôndrias/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/biossíntese , Ácido Pirúvico/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte da Membrana MitocondrialRESUMO
The pro-apoptotic BCL-2 protein BAX commits human cells to apoptosis by permeabilizing the outer mitochondrial membrane. BAX activation has been suggested to require the separation of helix α5 from α6 - the 'latch' from the 'core' domain - among other conformational changes. Here, we show that conformational changes in this region impair BAX translocation to the mitochondria and retrotranslocation back into the cytosol, and therefore BAX inhibition, but not activation. Redirecting misregulated BAX to the mitochondria revealed an alternative mechanism of BAX inhibition. The E3 ligase parkin, which is known to trigger mitochondria-specific autophagy, ubiquitylates BAX K128 and targets the pro-apoptotic BCL-2 protein for proteasomal degradation. Retrotranslocation-deficient BAX is completely degraded in a parkin-dependent manner. Although only a minor pool of endogenous BAX escapes retrotranslocation into the cytosol, parkin-dependent targeting of misregulated BAX on the mitochondria provides substantial protection against BAX apoptotic activity.
Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , Proteína X Associada a bcl-2/metabolismo , Apoptose , Citoproteção , Células HCT116 , Humanos , Lisina/metabolismo , Mitocôndrias/metabolismo , Estrutura Secundária de Proteína , Transporte Proteico , Ubiquitinação , Proteína X Associada a bcl-2/químicaRESUMO
The mitochondrial inner membrane harbors the complexes of the respiratory chain and translocase complexes for precursor proteins. We have identified a further subunit of the carrier translocase (TIM22 complex) that surprisingly is identical to subunit 3 of respiratory complex II, succinate dehydrogenase (Sdh3). The membrane-integral protein Sdh3 plays specific functions in electron transfer in complex II. We show by genetic and biochemical approaches that Sdh3 also plays specific functions in the TIM22 complex. Sdh3 forms a subcomplex with Tim18 and is involved in biogenesis and assembly of the membrane-integral subunits of the TIM22 complex. We conclude that the assembly of Sdh3 with different partner proteins, Sdh4 and Tim18, recruits it to two different mitochondrial membrane complexes with functions in bioenergetics and protein biogenesis, respectively.
Assuntos
Transporte de Elétrons , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Succinato Desidrogenase/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Membranas Mitocondriais/enzimologia , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologiaRESUMO
The elaborate membrane architecture of mitochondria is a prerequisite for efficient respiration and ATP generation. The cristae membranes, invaginations of the inner mitochondrial membrane, represent a specialized compartment that harbors the complexes of the respiratory chain and the F1Fo-ATP synthase. Crista junctions form narrow openings that connect the cristae membranes to the inner boundary membrane. The mitochondrial contact site and cristae organizing system (MICOS) is located at crista junctions where it stabilizes membrane curvature and forms contact sites between the mitochondrial inner and outer membranes. MICOS is a large machinery, consisting of two dynamic subcomplexes that are anchored in the inner membrane and expose domains to the intermembrane space. The functions of MICOS in mitochondrial membrane architecture and biogenesis are influenced by numerous interaction partners and the phospholipid environment.
Assuntos
DNA Mitocondrial/genética , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Membranas Mitocondriais/metabolismo , ATPases Translocadoras de Prótons/genética , Animais , DNA Mitocondrial/metabolismo , Regulação da Expressão Gênica , Humanos , Mitocôndrias/ultraestrutura , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/ultraestrutura , Fosfolipídeos/metabolismo , Ligação Proteica , ATPases Translocadoras de Prótons/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Especificidade da EspécieRESUMO
Mitochondria are multifunctional metabolic factories and integrative signaling organelles of eukaryotic cells. The structural basis for their numerous functions is a complex and dynamic double-membrane architecture. The outer membrane connects mitochondria to the cytosol and other organelles. The inner membrane is composed of a boundary region and highly folded cristae membranes. The evolutionarily conserved mitochondrial contact site and cristae organizing system (MICOS) connects the two inner membrane domains via formation and stabilization of crista junction structures. Moreover, MICOS establishes contact sites between inner and outer mitochondrial membranes by interacting with outer membrane protein complexes. MICOS deficiency leads to a grossly altered inner membrane architecture resulting in far-reaching functional perturbations in mitochondria. Consequently, mutations affecting the function of MICOS are responsible for a diverse spectrum of human diseases. In this article, we summarize recent insights and concepts on the role of MICOS in the organization of mitochondrial membranes. This article is part of a Special Issue entitled: Membrane Contact Sites edited by Christian Ungermann and Benoit Kornmann.
Assuntos
Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Animais , Humanos , Mitocôndrias/ultraestrutura , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Transdução de SinaisRESUMO
Mitochondrial F1Fo-ATP synthase generates the bulk of cellular ATP. This molecular machine assembles from nuclear- and mitochondria-encoded subunits. Whereas chaperones for formation of the matrix-exposed hexameric F1-ATPase core domain have been identified, insight into how the nuclear-encoded F1-domain assembles with the membrane-embedded Fo-region is lacking. Here we identified the INA complex (INAC) in the inner membrane of mitochondria as an assembly factor involved in this process. Ina22 and Ina17 are INAC constituents that physically associate with the F1-module and peripheral stalk, but not with the assembled F1Fo-ATP synthase. Our analyses show that loss of Ina22 and Ina17 specifically impairs formation of the peripheral stalk that connects the catalytic F1-module to the membrane embedded Fo-domain. We conclude that INAC represents a matrix-exposed inner membrane protein complex that facilitates peripheral stalk assembly and thus promotes a key step in the biogenesis of mitochondrial F1Fo-ATP synthase.
Assuntos
Membranas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
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.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/fisiologia , Transporte Proteico/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Autorradiografia , Eletroforese em Gel de Poliacrilamida , Proteínas de Membrana/química , Proteínas de Membrana Transportadoras/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Mutagênese , Reação em Cadeia da Polimerase , Estrutura Secundária de Proteína , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
The majority of mitochondrial proteins are synthesized with amino-terminal signal sequences. The presequence translocase of the inner membrane (TIM23 complex) mediates the import of these preproteins. The essential TIM23 core complex closely cooperates with partner protein complexes like the presequence translocase-associated import motor and the respiratory chain. The inner mitochondrial membrane also contains a large number of metabolite carriers, but their association with preprotein translocases has been controversial. We performed a comprehensive analysis of the TIM23 interactome based on stable isotope labeling with amino acids in cell culture. Subsequent biochemical studies on identified partner proteins showed that the mitochondrial ADP/ATP carrier associates with the membrane-embedded core of the TIM23 complex in a stoichiometric manner, revealing an unexpected connection of mitochondrial protein biogenesis to metabolite transport. Our data indicate that direct TIM23-AAC coupling may support preprotein import into mitochondria when respiratory activity is low.
Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/genética , Complexos Multiproteicos/genética , Transporte Proteico/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The yeast protein Zim17 belongs to a unique class of co-chaperones that maintain the solubility of Hsp70 proteins in mitochondria and plastids of eukaryotic cells. However, little is known about the functional cooperation between Zim17 and mitochondrial Hsp70 proteins in vivo. To analyze the effects of a loss of Zim17 function in the authentic environment, we introduced novel conditional mutations within the ZIM17 gene of the model organism Saccharomyces cerevisiae that allowed a recovery of temperature-sensitive but respiratory competent zim17 mutant cells. On fermentable growth medium, the mutant cells were prone to acquire respiratory deficits and showed a strong aggregation of the mitochondrial Hsp70 Ssq1 together with a concomitant defect in Fe/S protein biogenesis. In contrast, under respiring conditions, the mitochondrial Hsp70s Ssc1 and Ssq1 exhibited only a partial aggregation. We show that the induction of the zim17 mutant phenotype leads to strong import defects for Ssc1-dependent matrix-targeted precursor proteins that correlate with a significantly reduced binding of newly imported substrate proteins to Ssc1. We conclude that Zim17 is not only required for the maintenance of mtHsp70 solubility but also directly assists the functional interaction of mtHsp70 with substrate proteins in a J-type co-chaperone-dependent manner.