RESUMEN
BACKGROUND: Hydrogenosomes are a specific type of mitochondria that have adapted for life under anaerobiosis. Limited availability of oxygen has resulted in the loss of the membrane-associated respiratory chain, and consequently in the generation of minimal inner membrane potential (Δψ), and inefficient ATP synthesis via substrate-level phosphorylation. The changes in energy metabolism are directly linked with the organelle biogenesis. In mitochondria, proteins are imported across the outer membrane via the Translocase of the Outer Membrane (TOM complex), while two Translocases of the Inner Membrane, TIM22, and TIM23, facilitate import to the inner membrane and matrix. TIM23-mediated steps are entirely dependent on Δψ and ATP hydrolysis, while TIM22 requires only Δψ. The character of the hydrogenosomal inner membrane translocase and the mechanism of translocation is currently unknown. RESULTS: We report unprecedented modification of TIM in hydrogenosomes of the human parasite Trichomonas vaginalis (TvTIM). We show that the import of the presequence-containing protein into the hydrogenosomal matrix is mediated by the hybrid TIM22-TIM23 complex that includes three highly divergent core components, TvTim22, TvTim23, and TvTim17-like proteins. The hybrid character of the TvTIM is underlined by the presence of both TvTim22 and TvTim17/23, association with small Tim chaperones (Tim9-10), which in mitochondria are known to facilitate the transfer of substrates to the TIM22 complex, and the coupling with TIM23-specific ATP-dependent presequence translocase-associated motor (PAM). Interactome reconstruction based on co-immunoprecipitation (coIP) and mass spectrometry revealed that hybrid TvTIM is formed with the compositional variations of paralogs. Single-particle electron microscopy for the 132-kDa purified TvTIM revealed the presence of a single ring of small Tims complex, while mitochondrial TIM22 complex bears twin small Tims hexamer. TvTIM is currently the only TIM visualized outside of Opisthokonta, which raised the question of which form is prevailing across eukaryotes. The tight association of the hybrid TvTIM with ADP/ATP carriers (AAC) suggests that AAC may directly supply ATP for the protein import since ATP synthesis is limited in hydrogenosomes. CONCLUSIONS: The hybrid TvTIM in hydrogenosomes represents an original structural solution that evolved for protein import when Δψ is negligible and remarkable example of evolutionary adaptation to an anaerobic lifestyle.
Asunto(s)
Transporte de Proteínas , Trichomonas vaginalis , Trichomonas vaginalis/metabolismo , Proteínas Protozoarias/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Mitocondrias/metabolismo , Orgánulos/metabolismoRESUMEN
TIMM50 is a core subunit of the TIM23 complex, the mitochondrial inner membrane translocase responsible for the import of pre-sequence-containing precursors into the mitochondrial matrix and inner membrane. Here we describe a mitochondrial disease patient who is homozygous for a novel variant in TIMM50 and establish the first proteomic map of mitochondrial disease associated with TIMM50 dysfunction. We demonstrate that TIMM50 pathogenic variants reduce the levels and activity of endogenous TIM23 complex, which significantly impacts the mitochondrial proteome, resulting in a combined oxidative phosphorylation (OXPHOS) defect and changes to mitochondrial ultrastructure. Using proteomic data sets from TIMM50 patient fibroblasts and a TIMM50 HEK293 cell model of disease, we reveal that laterally released substrates imported via the TIM23SORT complex pathway are most sensitive to loss of TIMM50. Proteins involved in OXPHOS and mitochondrial ultrastructure are enriched in the TIM23SORT substrate pool, providing a biochemical mechanism for the specific defects in TIMM50-associated mitochondrial disease patients. These results highlight the power of using proteomics to elucidate molecular mechanisms of disease and uncovering novel features of fundamental biology, with the implication that human TIMM50 may have a more pronounced role in lateral insertion than previously understood.
Asunto(s)
Mitocondrias , Enfermedades Mitocondriales , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Fosforilación Oxidativa , Transporte de Proteínas , Humanos , Fibroblastos/metabolismo , Células HEK293 , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/genética , Mitocondrias/metabolismo , Enfermedades Mitocondriales/metabolismo , Enfermedades Mitocondriales/patología , Enfermedades Mitocondriales/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/genética , Membranas Mitocondriales/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Mutación/genética , Proteómica/métodosRESUMEN
The majority of mitochondrial proteins are encoded in the nucleus, translated on cytosolic ribosomes, and subsequently targeted to the mitochondrial surface. Their further import into the organelle is facilitated by highly specialized protein translocases. Mitochondrial precursor proteins that are destined to the mitochondrial matrix and, to some extent, the inner membrane, utilize translocase of the inner membrane (TIM23). This indispensable import machinery has been extensively studied in yeast. The translocating unit of the TIM23 complex in yeast consists of two membrane proteins, Tim17 and Tim23. In contrast to previous findings, recent reports demonstrate the primary role of Tim17, rather than Tim23, in the translocation of newly synthesized proteins. Very little is known about human TIM23 translocase. Human cells have two orthologs of yeast Tim17, TIMM17A and TIMM17B. Here, using computational tools, we present the architecture of human core TIM23 variants with either TIMM17A or TIMM17B, forming two populations of highly similar complexes. The structures reveal high conservation of the core TIM23 complex between human and yeast. Interestingly, both TIMM17A and TIMM17B variants interact with TIMM23 and reactive oxygen species modulator 1 (ROMO1); a homolog of yeast Mgr2, a protein that can create a channel-like structure with Tim17. The high structural conservation of proteins that form the core TIM23 complex in yeast and humans raises an interesting question about mechanistic and functional differences that justify existence of the two variants of TIM23 in higher eukaryotes.
RESUMEN
The inner membrane of mitochondria contains hundreds of different integral membrane proteins. These proteins transport molecules into and out of the matrix, they carry out multifold catalytic reactions and they promote the biogenesis or degradation of mitochondrial constituents. Most inner membrane proteins are encoded by nuclear genes and synthesized in the cytosol from where they are imported into mitochondria by translocases in the outer and inner membrane. Three different import routes direct proteins into the inner membrane and allow them to acquire their appropriate membrane topology. First, mitochondrial import intermediates can be arrested at the level of the TIM23 inner membrane translocase by a stop-transfer sequence to reach the inner membrane by lateral insertion. Second, proteins can be fully translocated through the TIM23 complex into the matrix from where they insert into the inner membrane in an export-like reaction. Carriers and other polytopic membrane proteins embark on a third insertion pathway: these hydrophobic proteins employ the specialized TIM22 translocase to insert from the intermembrane space (IMS) into the inner membrane. This review article describes these three targeting routes and provides an overview of the machinery that promotes the topogenesis of mitochondrial inner membrane proteins.
RESUMEN
Most mitochondrial proteins are nuclear-encoded and imported by the protein import machinery based on specific targeting signals. The proteins that carry an amino-terminal targeting signal (presequence) are imported via the presequence import pathway that involves the translocases of the outer and inner membranes - TOM and TIM23 complexes. In this article, we discuss how mitochondrial matrix and inner membrane precursor proteins are imported along the presequence pathway in Saccharomyces cerevisiae with a focus on the dynamics of the TIM23 complex, and further update with some of the key findings that advanced the field in the last few years.
Asunto(s)
Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales , Transporte de Proteínas , Saccharomyces cerevisiae , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Transporte de Membrana , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas de Saccharomyces cerevisiae , Proteínas Portadoras/metabolismo , Transporte de Electrón , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales/metabolismo , Transporte de Proteínas , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Mitochondria are essential organelles for cellular energy production, metabolic homeostasis, calcium homeostasis, cell proliferation, and apoptosis. About 99% of mammalian mitochondrial proteins are encoded by the nuclear genome, synthesized as precursors in the cytosol, and imported into mitochondria by mitochondrial protein import machinery. Mitochondrial protein import systems function not only as independent units for protein translocation, but also are deeply integrated into a functional network of mitochondrial bioenergetics, protein quality control, mitochondrial dynamics and morphology, and interaction with other organelles. Mitochondrial protein import deficiency is linked to various diseases, including cardiovascular disease. In this review, we describe an emerging class of protein or genetic variations of components of the mitochondrial import machinery involved in heart disease. The major protein import pathways, including the presequence pathway (TIM23 pathway), the carrier pathway (TIM22 pathway), and the mitochondrial intermembrane space import and assembly machinery, related translocases, proteinases, and chaperones, are discussed here. This review highlights the importance of mitochondrial import machinery in heart disease, which deserves considerable attention, and further studies are urgently needed. Ultimately, this knowledge may be critical for the development of therapeutic strategies in heart disease.
RESUMEN
Mitochondria import about 1000 proteins that are produced as precursors on cytosolic ribosomes. Defects in mitochondrial protein import result in the accumulation of non-imported precursor proteins and proteotoxic stress. The cell is equipped with different quality control mechanisms to monitor protein transport into mitochondria. First, molecular chaperones guide unfolded proteins to mitochondria and deliver non-imported proteins to proteasomal degradation. Second, quality control factors remove translocation stalled precursor proteins from protein translocases. Third, protein translocases monitor protein sorting to mitochondrial subcompartments. Fourth, AAA proteases of the mitochondrial subcompartments remove mislocalized or unassembled proteins. Finally, impaired efficiency of protein transport is an important sensor for mitochondrial dysfunction and causes the induction of cellular stress responses, which could eventually result in the removal of the defective mitochondria by mitophagy. In this review, we summarize our current understanding of quality control mechanisms that govern mitochondrial protein transport.
Asunto(s)
Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Membranas Mitocondriales/metabolismo , Mitofagia , Precursores de Proteínas/metabolismo , Animales , Humanos , Modelos Biológicos , Transporte de Proteínas , Control de CalidadRESUMEN
The vast majority of mitochondrial proteins are encoded in the nuclear genome and synthesized on cytosolic ribosomes as precursor proteins with specific mitochondrial targeting signals. Mitochondrial targeting signals are very diverse, however, about 70% of mitochondrial proteins carry cleavable, N-terminal extensions called presequences. These amphipathic helices with one positively charged and one hydrophobic surface target proteins to the mitochondrial matrix with the help of the TOM and TIM23 complexes in the outer and inner membranes, respectively. Translocation of proteins across the two mitochondrial membranes does not take place independently of each other. Rather, in the intermembrane space, where the two complexes meet, components of the TOM and TIM23 complexes form an intricate network of protein-protein interactions that mediates initially transfer of presequences and then of the entire precursor proteins from the outer to the inner mitochondrial membrane. In this Mini Review, we summarize our current understanding of how the TOM and TIM23 complexes cooperate with each other and highlight some of the future challenges and unresolved questions in the field.
RESUMEN
In the intermembrane space (IMS) of mitochondria, the receptor domain of Tim23 has an essential role during translocation of hundreds of different proteins from the cytosol via the TOM and TIM23 complexes in the outer and inner membranes, respectively. This intrinsically disordered domain, which can even extend into the cytosol, was shown, mostly in vitro, to interact with several subunits of the TOM and TIM23 complexes. To obtain molecular understanding of this organizational hub in the IMS, we dissected the IMS domain of Tim23 in vivo. We show that the interaction surface of Tim23 with Tim50 is larger than previously thought and reveal an unexpected interaction of Tim23 with Pam17 in the IMS, impairment of which influences their interaction in the matrix. Furthermore, mutations of two conserved negatively charged residues of Tim23, close to the inner membrane, prevented dimerization of Tim23. The same mutations increased exposure of Tim23 on the mitochondrial surface, whereas dissipation of membrane potential decreased it. Our results reveal an intricate network of Tim23 interactions in the IMS, whose influence is transduced across two mitochondrial membranes, ensuring efficient translocation of proteins into mitochondria.
Asunto(s)
Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/química , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Mutación , Sitios de Unión , Proteínas de Transporte de Membrana Mitocondrial/genética , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Transporte de Proteínas , Desplegamiento ProteicoRESUMEN
Mitochondria are deeply integrated into crucial functions of eukaryotic cells, including ATP production via oxidative phosphorylation, biosynthesis of iron-sulfur clusters, amino acids, lipids and heme, signaling pathways, and programmed cell death. The import of about 1000 different proteins that are produced as precursors on cytosolic ribosomes is essential for mitochondrial functions and biogenesis. The translocase of the outer mitochondrial membrane (TOM) forms the entry gate for the vast majority of mitochondrial proteins. Research of the last years has uncovered a complicated network of protein translocases and pathways that sort proteins into the mitochondrial subcompartments: outer and inner membranes, intermembrane space, and matrix. The in vitro import of a large number of different precursor proteins into mitochondria has been a pivotal experimental assay to identify these protein-sorting routes. This experimental set-up enables studies on the kinetics of protein transport into isolated mitochondria, on the processing of precursor proteins, and on their assembly into functional protein machineries. In vitro protein import assays are widely used and are indispensable for research on mitochondrial protein biogenesis.
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Técnicas Citológicas/métodos , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Animales , Sistema Libre de Células , Reactivos de Enlaces Cruzados/química , Disulfuros/metabolismo , Humanos , Péptido Hidrolasas/metabolismo , Transporte de Proteínas , Conejos , Saccharomyces cerevisiae/metabolismo , SolubilidadRESUMEN
Mitochondrial precursor proteins with amino-terminal presequences are imported via the presequence pathway, utilizing the TIM23 complex for inner membrane translocation. Initially, the precursors pass the outer membrane through the TOM complex and are handed over to the TIM23 complex where they are sorted into the inner membrane or translocated into the matrix. This handover process depends on the receptor proteins at the inner membrane, Tim50 and Tim23, which are critical for efficient import. In this review, we summarize key findings that shaped the current concepts of protein translocation along the presequence import pathway, with a particular focus on the precursor handover process from TOM to the TIM23 complex. In addition, we discuss functions of the human TIM23 pathway and the recently uncovered pathogenic mutations in TIM50.
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Proteínas Portadoras/metabolismo , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Humanos , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Transporte de ProteínasRESUMEN
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.
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Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Membranas Mitocondriales/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte BiológicoRESUMEN
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.
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Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/metabolismo , Mitocondrias/enzimología , Membranas Mitocondriales/enzimología , Proteínas Mitocondriales/metabolismo , Enfermedad de Parkinson/enzimología , Proteínas Quinasas/metabolismo , Secuencias de Aminoácidos , Antiparkinsonianos/farmacología , Transporte Biológico , Diseño de Fármacos , Activación Enzimática , Células HeLa , Humanos , Potencial de la Membrana Mitocondrial , Proteínas de la Membrana/genética , Metaloendopeptidasas/genética , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Membranas Mitocondriales/efectos de los fármacos , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales/genética , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/genética , Dominios y Motivos de Interacción de Proteínas , Proteínas Quinasas/genética , Proteolisis , Transducción de Señal , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
BACKGROUND: The existence of mitochondria-related organelles (MROs) is proposed for eukaryotic organisms. The Amoebozoa includes some organisms that are known to have mitosomes but also organisms that have aerobic mitochondria. However, the mitochondrial protein apparatus of this supergroup remains largely unsampled, except for the mitochondrial outer membrane import complexes studied recently. Therefore, in this study we investigated the mitochondrial inner membrane and intermembrane space complexes, using the available genome and transcriptome sequences. RESULTS: When compared with the canonical cognate complexes described for the yeast Saccharomyces cerevisiae, amoebozoans with aerobic mitochondria, display lower differences in the number of subunits predicted for these complexes than the mitochondrial outer membrane complexes, although the predicted subunits appear to display different levels of diversity in regard to phylogenetic position and isoform numbers. For the putative mitosome-bearing amoebozoans, the number of predicted subunits suggests the complex elimination distinctly more pronounced than in the case of the outer membrane ones. CONCLUSION: The results concern the problem of mitochondrial and mitosome protein import machinery structural variability and the reduction of their complexity within the currently defined supergroup of Amoebozoa. This results are crucial for better understanding of the Amoebozoa taxa of both biomedical and evolutionary importance.
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Amebozoos/genética , Proteínas de Transporte de Membrana Mitocondrial/genética , Acanthamoeba castellanii/genética , Amebozoos/clasificación , Células Cultivadas , Dictyostelium/genética , Perfilación de la Expresión Génica , Genómica , Proteínas de Transporte de Membrana Mitocondrial/clasificación , Filogenia , Subunidades de Proteína/genéticaRESUMEN
Many mitochondrial proteins perform their functions as components of large, multimeric complexes. Chemical crosslinking is a powerful method to analyze protein-protein interactions within such complexes. Using membrane-permeable crosslinkers and isolated intact mitochondria, protein-protein interactions that are secluded by two mitochondrial membranes can be readily analyzed in physiologically active, isolated organelles under a variety of physiological and pathophysiological conditions. Here, we describe two methods for chemical crosslinking in intact yeast mitochondria. The first method enables the analysis of ATP-dependent remodeling of mitochondrial protein complexes while the second one allows the identification of crosslinking partners of a protein of interest.
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Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Mapeo de Interacción de Proteínas/métodos , Adenosina Trifosfato/metabolismo , Reactivos de Enlaces Cruzados , Proteínas Mitocondriales/química , Proteínas Mitocondriales/aislamiento & purificación , Complejos Multiproteicos/metabolismo , Unión ProteicaRESUMEN
The majority of mitochondrial proteins use N-terminal presequences for targeting to mitochondria and are translocated by the presequence translocase. During translocation, proteins, threaded through the channel in the inner membrane, are handed over to the import motor at the matrix face. Tim17 is an essential, membrane-embedded subunit of the translocase; however, its function is only poorly understood. Here, we functionally dissected its four predicted transmembrane (TM) segments. Mutations in TM1 and TM2 impaired the interaction of Tim17 with Tim23, component of the translocation channel, whereas mutations in TM3 compromised binding of the import motor. We identified residues in the matrix-facing region of Tim17 involved in binding of the import motor. Our results reveal functionally distinct roles of different regions of Tim17 and suggest how they may be involved in handing over the proteins, during their translocation into mitochondria, from the channel to the import motor of the presequence translocase.
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Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas Mutantes/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Análisis Mutacional de ADN , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Modelos Biológicos , Modelos Químicos , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Mitochondria acquire the majority of their proteins from the cytosol in a process that is mediated by intricate multimeric machineries designed to allow proteins to cross and/or to insert themselves into the two mitochondrial membranes. Ongoing studies carried out in yeast over the past few decades have led to the discovery of numerous protein components that constitute several mitochondrial translocases. One of these complexes, the mitochondrial TIM23, is the major translocase for matrix proteins and is the focus of this review. The components of the TIM23 complex are categorized into four functional types. The first type plays the role of receptor for preproteins in the intermembrane space. The second type forms the actual channel that allows proteins to cross the inner mitochondrial membrane. The third species functions as part of the motor that mediates the final steps of import across the inner membrane. Additional components play regulatory roles orchestrating the action of this myriad of subunits. Recent studies provide new insights into the function of the mammalian TIM23 complex and the role that it plays under pathological conditions.
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Proteínas Mitocondriales/metabolismo , Animales , Enfermedad , Humanos , Membranas Mitocondriales/metabolismo , Modelos Biológicos , Transporte de ProteínasRESUMEN
Two protein translocases transport precursor proteins into or across the inner mitochondrial membrane. The presequence translocase (TIM23 complex) sorts precursor proteins with a cleavable presequence either into the matrix or into the inner membrane. The carrier translocase (TIM22 complex) inserts multispanning proteins into the inner membrane. Both protein import pathways depend on the presence of a membrane potential, which is generated by the activity of the respiratory chain. The non-bilayer-forming phospholipids cardiolipin and phosphatidylethanolamine are required for the activity of the respiratory chain and therefore to maintain the membrane potential for protein import. Depletion of cardiolipin further affects the stability of the TIM23 complex. The role of bilayer-forming phospholipids like phosphatidylcholine (PC) in protein transport into the inner membrane and the matrix is unknown. Here, we report that import of presequence-containing precursors and carrier proteins is impaired in PC-deficient mitochondria. Surprisingly, depletion of PC does not affect stability and activity of respiratory supercomplexes, and the membrane potential is maintained. Instead, the dynamic TIM23 complex is destabilized when the PC levels are reduced, whereas the TIM22 complex remains intact. Our analysis further revealed that initial precursor binding to the TIM23 complex is impaired in PC-deficient mitochondria. We conclude that reduced PC levels differentially affect the TIM22 and TIM23 complexes in mitochondrial protein transport.
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Proteínas de Transporte de Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Membranas Mitocondriales/metabolismo , Fosfatidilcolinas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte de Membrana/genética , Mitocondrias/genética , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Fosfatidilcolinas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The preprotein translocase of the inner membrane of mitochondria (TIM23 complex) is the main entry gate for proteins of the matrix and the inner membrane. Tim23p, the core component of TIM23 complex, forms the import pore across the inner membrane and exerts a key function in the protein import. However, the interaction of divalent metal ions with Tim23p and the contribution in the interaction of presequence peptide with Tim23p are still unknown. Herein, we investigated the interaction of divalent metal ions with the intermembrane space domain of Tim23p (Tim23IMS) and the interaction of presequence peptides with Tim23IMS in presence of Ca(2+) ion by fluorescence spectroscopy in vitro. The static fluorescence quenching indicates the existence of strong binding between divalent metal ions and Tim23IMS. The order of the binding strength is Ca(2+), Mg(2+), Cu(2+), Mn(2+), and Co(2+) (from strong to weak). Moreover, the interaction of presequence peptides with Tim23IMS is weakened in presence of Ca(2+) ion, which implicates that Ca(2+) ion may play an important role in the protein import by TIM23 complex.