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1.
J Cell Sci ; 136(10)2023 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-37073556

RESUMO

Mitochondria are essential organelles of eukaryotic cells and are characterized by their unique and complex membrane system. They are confined from the cytosol by an envelope consisting of two membranes. Signals, metabolites, proteins and lipids have to be transferred across these membranes via proteinaceous contact sites to keep mitochondria functional. In the present study, we identified a novel mitochondrial contact site in Saccharomyces cerevisiae that is formed by the inner membrane protein Cqd1 and the outer membrane proteins Por1 and Om14. Similar to what is found for the mitochondrial porin Por1, Cqd1 is highly conserved, suggesting that this complex is conserved in form and function from yeast to human. Cqd1 is a member of the UbiB protein kinase-like family (also called aarF domain-containing kinases). It was recently shown that Cqd1, in cooperation with Cqd2, controls the cellular distribution of coenzyme Q by a yet unknown mechanism. Our data suggest that Cqd1 is additionally involved in phospholipid homeostasis. Moreover, overexpression of CQD1 and CQD2 causes tethering of mitochondria to the endoplasmic reticulum, which might explain the ability of Cqd2 to rescue ERMES deletion phenotypes.


Assuntos
Mitocôndrias , Proteínas de Saccharomyces cerevisiae , Humanos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo
2.
Nat Struct Mol Biol ; 27(2): 142-149, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31988523

RESUMO

Some proteins require completion of folding before translocation across a membrane into another cellular compartment. Yet the permeability barrier of the membrane should not be compromised and mechanisms have remained mostly elusive. Here, we present the structure of Saccharomyces cerevisiae Bcs1, an AAA-ATPase of the inner mitochondrial membrane. Bcs1 facilitates the translocation of the Rieske protein, Rip1, which requires folding and incorporation of a 2Fe-2S cluster before translocation and subsequent integration into the bc1 complex. Surprisingly, Bcs1 assembles into exclusively heptameric homo-oligomers, with each protomer consisting of an amphipathic transmembrane helix, a middle domain and an ATPase domain. Together they form two aqueous vestibules, the first being accessible from the mitochondrial matrix and the second positioned in the inner membrane, with both separated by the seal-forming middle domain. On the basis of this unique architecture, we propose an airlock-like translocation mechanism for folded Rip1.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas Mitocondriais/metabolismo , Chaperonas Moleculares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , ATPases Associadas a Diversas Atividades Celulares/química , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Membranas Mitocondriais/química , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/química , Modelos Moleculares , Chaperonas Moleculares/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Conformação Proteica , Domínios Proteicos , Dobramento de Proteína , Multimerização Proteica , Transporte Proteico , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química
3.
Nature ; 571(7764): E4, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31235950

RESUMO

Change history: In this Letter, the bottom blot in Fig. 2g (for 'IB: Myc') was missing. This has been corrected online.

4.
Nature ; 570(7762): 538-542, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31189955

RESUMO

Ribosome-associated quality control (RQC) provides a rescue pathway for eukaryotic cells to process faulty proteins after translational stalling of cytoplasmic ribosomes1-6. After dissociation of ribosomes, the stalled tRNA-bound peptide remains associated with the 60S subunit and extended by Rqc2 by addition of C-terminal alanyl and threonyl residues (CAT tails)7-9, whereas Vms1 catalyses cleavage and release of the peptidyl-tRNA before or after addition of CAT tails10-12. In doing so, Vms1 counteracts CAT-tailing of nuclear-encoded mitochondrial proteins that otherwise drive aggregation and compromise mitochondrial and cellular homeostasis13. Here we present structural and functional insights into the interaction of Saccharomyces cerevisiae Vms1 with 60S subunits in pre- and post-peptidyl-tRNA cleavage states. Vms1 binds to 60S subunits with its Vms1-like release factor 1 (VLRF1), zinc finger and ankyrin domains. VLRF1 overlaps with the Rqc2 A-tRNA position and interacts with the ribosomal A-site, projecting its catalytic GSQ motif towards the CCA end of the tRNA, its Y285 residue dislodging the tRNA A73 for nucleolytic cleavage. Moreover, in the pre-state, we found the ABCF-type ATPase Arb1 in the ribosomal E-site, which stabilizes the delocalized A73 of the peptidyl-tRNA and stimulates Vms1-dependent tRNA cleavage. Our structural analysis provides mechanistic insights into the interplay of the RQC factors Vms1, Rqc2 and Arb1 and their role in the protection of mitochondria from the aggregation of toxic proteins.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Homeostase , Proteínas Mitocondriais/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/ultraestrutura , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/ultraestrutura , Sequência de Aminoácidos , Proteínas de Transporte/ultraestrutura , Microscopia Crioeletrônica , Modelos Moleculares , Proteoma/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/química , Subunidades Ribossômicas Maiores de Eucariotos/genética , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Ribossomos/química , Ribossomos/genética , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestrutura
5.
Sci Rep ; 9(1): 2012, 2019 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-30765764

RESUMO

Maintenance of the mitochondrial proteome depends on import of newly made proteins from the cytosol. More than half of mitochondrial proteins are made as precursor proteins with N-terminal extensions called presequences and use the TIM23 complex for translocation into the matrix, the inner mitochondrial membrane and the intermembrane space (IMS). Tim50 is the central receptor of the complex that recognizes precursor proteins in the IMS. Additionally, Tim50 interacts with the IMS domain of the channel forming subunit, Tim23, an interaction that is essential for protein import across the mitochondrial inner membrane. In order to gain deeper insight into the molecular function of Tim50, we used random mutagenesis to determine residues that are important for its function. The temperature-sensitive mutants isolated were defective in import of TIM23-dependent precursor proteins. The residues mutated map to two distinct patches on the surface of Tim50. Notably, mutations in both patches impaired the interaction of Tim50 with Tim23. We propose that two regions of Tim50 play a role in its interaction with Tim23 and thereby affect the import function of the complex.


Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Mutagênese , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/química , Temperatura
6.
Cell ; 171(4): 890-903.e18, 2017 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-29107329

RESUMO

Eukaryotic cells have evolved extensive protein quality-control mechanisms to remove faulty translation products. Here, we show that yeast cells continually produce faulty mitochondrial polypeptides that stall on the ribosome during translation but are imported into the mitochondria. The cytosolic protein Vms1, together with the E3 ligase Ltn1, protects against the mitochondrial toxicity of these proteins and maintains cell viability under respiratory conditions. In the absence of these factors, stalled polypeptides aggregate after import and sequester critical mitochondrial chaperone and translation machinery. Aggregation depends on C-terminal alanyl/threonyl sequences (CAT-tails) that are attached to stalled polypeptides on 60S ribosomes by Rqc2. Vms1 binds to 60S ribosomes at the mitochondrial surface and antagonizes Rqc2, thereby facilitating import, impeding aggregation, and directing aberrant polypeptides to intra-mitochondrial quality control. Vms1 is a key component of a rescue pathway for ribosome-stalled mitochondrial polypeptides that are inaccessible to ubiquitylation due to coupling of translation and translocation.


Assuntos
Proteínas de Transporte/metabolismo , Mitocôndrias/fisiologia , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Citosol/metabolismo , Transporte de Elétrons , Homeostase , Saccharomyces cerevisiae/fisiologia , Ubiquitina-Proteína Ligases/metabolismo
7.
Methods Mol Biol ; 1567: 293-314, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28276026

RESUMO

Budding yeast Saccharomyces cerevisiae represents a widely used model organism for the study of mitochondrial biogenesis and architecture. Electron microscopy is an essential tool in the analysis of cellular ultrastructure and the precise localization of proteins to organellar subcompartments. We provide here detailed protocols for the analysis of yeast mitochondria by transmission electron microscopy: (1) chemical fixation and Epon embedding of yeast cells and isolated mitochondria, and (2) cryosectioning and immunolabeling of yeast cells and isolated mitochondria according to the Tokuyasu method.


Assuntos
Microscopia Eletrônica , Mitocôndrias/ultraestrutura , Leveduras/ultraestrutura , Crioultramicrotomia/métodos , Microscopia Eletrônica/métodos , Microscopia Eletrônica de Transmissão , Microscopia Imunoeletrônica , Organelas/ultraestrutura , Saccharomyces cerevisiae/ultraestrutura , Fluxo de Trabalho
8.
Elife ; 62017 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-28165323

RESUMO

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.


Assuntos
Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mutantes/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Análise Mutacional de DNA , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Modelos Biológicos , Modelos Químicos , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas de Saccharomyces cerevisiae/genética
9.
Elife ; 52016 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-27849155

RESUMO

Metabolic function and architecture of mitochondria are intimately linked. More than 60 years ago, cristae were discovered as characteristic elements of mitochondria that harbor the protein complexes of oxidative phosphorylation, but how cristae are formed, remained an open question. Here we present experimental results obtained with yeast that support a novel hypothesis on the existence of two molecular pathways that lead to the generation of lamellar and tubular cristae. Formation of lamellar cristae depends on the mitochondrial fusion machinery through a pathway that is required also for homeostasis of mitochondria and mitochondrial DNA. Tubular cristae are formed via invaginations of the inner boundary membrane by a pathway independent of the fusion machinery. Dimerization of the F1FO-ATP synthase and the presence of the MICOS complex are necessary for both pathways. The proposed hypothesis is suggested to apply also to higher eukaryotes, since the key components are conserved in structure and function throughout evolution.


Assuntos
GTP Fosfo-Hidrolases/genética , Proteínas de Ligação ao GTP/genética , Mitocôndrias/genética , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Expressão Gênica , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Dinâmica Mitocondrial/fisiologia , Membranas Mitocondriais/ultraestrutura , Proteínas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Biogênese de Organelas , Multimerização Proteica , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo
12.
Genome Biol Evol ; 7(5): 1235-51, 2015 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-25861818

RESUMO

The five macromolecular complexes that jointly mediate oxidative phosphorylation (OXPHOS) in mitochondria consist of many more subunits than those of bacteria, yet, it remains unclear by which evolutionary mechanism(s) these novel subunits were recruited. Even less well understood is the structural evolution of mitochondrial ribosomes (mitoribosomes): while it was long thought that their exceptionally high protein content would physically compensate for their uniquely low amount of ribosomal RNA (rRNA), this hypothesis has been refuted by structural studies. Here, we present a cryo-electron microscopy structure of the 73S mitoribosome from Neurospora crassa, together with genomic and proteomic analyses of mitoribosome composition across the eukaryotic domain. Surprisingly, our findings reveal that both structurally and compositionally, mitoribosomes have evolved very similarly to mitochondrial OXPHOS complexes via two distinct phases: A constructive phase that mainly acted early in eukaryote evolution, resulting in the recruitment of altogether approximately 75 novel subunits, and a reductive phase that acted during metazoan evolution, resulting in gradual length-reduction of mitochondrially encoded rRNAs and OXPHOS proteins. Both phases can be well explained by the accumulation of (slightly) deleterious mutations and deletions, respectively, in mitochondrially encoded rRNAs and OXPHOS proteins. We argue that the main role of the newly recruited (nuclear encoded) ribosomal- and OXPHOS proteins is to provide structural compensation to the mutationally destabilized mitochondrially encoded components. While the newly recruited proteins probably provide a selective advantage owing to their compensatory nature, and while their presence may have opened evolutionary pathways toward novel mitochondrion-specific functions, we emphasize that the initial events that resulted in their recruitment was nonadaptive in nature. Our framework is supported by population genetic studies, and it can explain the complete structural evolution of mitochondrial ribosomes and OXPHOS complexes, as well as many observed functions of individual proteins.


Assuntos
Evolução Molecular , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Ribossomos/química , Genes Mitocondriais , Mutação , Neurospora crassa/genética , Fosforilação Oxidativa , Subunidades Proteicas/genética , RNA Ribossômico/química , Proteínas Ribossômicas/química , Ribossomos/ultraestrutura
13.
FEBS J ; 282(11): 2178-86, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25765297

RESUMO

Approximately 99% of the mitochondrial proteome is nucleus-encoded, synthesized in the cytosol, and subsequently imported into and sorted to the correct compartment in the organelle. The translocase of the inner mitochondrial membrane 23 (TIM23) complex is the major protein translocase of the inner membrane, and is responsible for translocation of proteins across the inner membrane and their insertion into the inner membrane. Tim23 is the central component of the complex that forms the import channel. A high-resolution structure of the import channel is still missing, and structural elements important for its function are unknown. In the present study, we analyzed the importance of the highly abundant GxxxG motifs in the transmembrane segments of Tim23 for the structural integrity of the TIM23 complex. Of 10 glycines present in the GxxxG motifs in the first, second and third transmembrane segments of Tim23, mutations of three of them in transmembrane segments 1 and 2 resulted in a lethal phenotype, and mutations of three others in a temperature-sensitive phenotype. The remaining four caused no obvious growth phenotype. Importantly, none of the mutations impaired the import and membrane integration of Tim23 precursor into mitochondria. However, the severity of growth impairment correlated with the destabilization of the TIM23 complex. We conclude that the GxxxG motifs found in the first and second transmembrane segments of Tim23 are necessary for the structural integrity of the TIM23 complex.


Assuntos
Proteínas de Membrana Transportadoras/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Motivos de Aminoácidos , Proteínas de Membrana Transportadoras/química , Mitocôndrias/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Ligação Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Transporte Proteico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química
14.
J Mol Biol ; 427(6 Pt A): 1135-58, 2015 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-25676309

RESUMO

Mitochondria are the central hub of key cellular processes such as energy conversion, cell signaling, cell cycle regulation and cell differentiation. Therefore, in particular, mitochondrial biogenesis and protein translocation have been the focus of intense research for now nearly half a century. In spite of remarkable progress the field has made, many of the proposed mechanisms remain controversial and none of the translocation pathways is yet understood at the high-resolution level. In this context, the present article is intended to identify and discuss current major open questions and unresolved issues in the field in hope that it will stimulate and engage the pursuit of current efforts and expose new directions.


Assuntos
Proteínas de Transporte/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , Citosol/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Complexos Multiproteicos/metabolismo , Precursores de Proteínas/metabolismo , Transporte Proteico , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Supressoras de Tumor/metabolismo
15.
J Mol Biol ; 427(5): 1075-84, 2015 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-25083920

RESUMO

Translocation of the majority of mitochondrial proteins from the cytosol into mitochondria requires the cooperation of TOM and TIM23 complexes in the outer and inner mitochondrial membranes. The molecular mechanisms underlying this cooperation remain largely unknown. Here, we present biochemical and genetic evidence that at least two contacts from the side of the TIM23 complex play an important role in TOM-TIM23 cooperation in vivo. Tim50, likely through its very C-terminal segment, interacts with Tom22. This interaction is stimulated by translocating proteins and is independent of any other TOM-TIM23 contact known so far. Furthermore, the exposure of Tim23 on the mitochondrial surface depends not only on its interaction with Tim50 but also on the dynamics of the TOM complex. Destabilization of the individual contacts reduces the efficiency of import of proteins into mitochondria and destabilization of both contacts simultaneously is not tolerated by yeast cells. We conclude that an intricate and coordinated network of protein-protein interactions involving primarily Tim50 and also Tim23 is required for efficient translocation of proteins across both mitochondrial membranes.


Assuntos
Proteínas de Transporte/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Transporte Proteico/fisiologia , Proteínas Fúngicas/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Domínios e Motivos de Interação entre Proteínas/fisiologia , Leveduras/metabolismo
16.
Elife ; 3: e01684, 2014 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-24714493

RESUMO

Structure and function of mitochondria are intimately linked. In a search for components that participate in building the elaborate architecture of this complex organelle we have identified Aim24, an inner membrane protein. Aim24 interacts with the MICOS complex that is required for the formation of crista junctions and contact sites between inner and outer membranes. Aim24 is necessary for the integrity of the MICOS complex, for normal respiratory growth and mitochondrial ultrastructure. Modification of MICOS subunits Mic12 or Mic26 by His-tags in the absence of Aim24 leads to complete loss of cristae and respiratory complexes. In addition, the level of tafazzin, a cardiolipin transacylase, is drastically reduced and the composition of cardiolipin is modified like in mutants lacking tafazzin. In conclusion, Aim24 by interacting with the MICOS complex plays a key role in mitochondrial architecture, composition and function. DOI: http://dx.doi.org/10.7554/eLife.01684.001.


Assuntos
Cardiolipinas/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Biogênese de Organelas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Oxirredução , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo
17.
J Cell Biol ; 204(7): 1083-6, 2014 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-24687277

RESUMO

The mitochondrial inner membrane contains a large protein complex that functions in inner membrane organization and formation of membrane contact sites. The complex was variably named the mitochondrial contact site complex, mitochondrial inner membrane organizing system, mitochondrial organizing structure, or Mitofilin/Fcj1 complex. To facilitate future studies, we propose to unify the nomenclature and term the complex "mitochondrial contact site and cristae organizing system" and its subunits Mic10 to Mic60.


Assuntos
Membranas Mitocondriais/química , Proteínas Mitocondriais/química , Subunidades Proteicas/química , Animais , Humanos , Modelos Moleculares , Complexos Multiproteicos/química , Terminologia como Assunto
18.
J Cell Biol ; 199(4): 599-611, 2012 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-23128244

RESUMO

The TOB-SAM complex is an essential component of the mitochondrial outer membrane that mediates the insertion of ß-barrel precursor proteins into the membrane. We report here its isolation and determine its size, composition, and structural organization. The complex from Neurospora crassa was composed of Tob55-Sam50, Tob38-Sam35, and Tob37-Sam37 in a stoichiometry of 1:1:1 and had a molecular mass of 140 kD. A very minor fraction of the purified complex was associated with one Mdm10 protein. Using molecular homology modeling for Tob55 and cryoelectron microscopy reconstructions of the TOB complex, we present a model of the TOB-SAM complex that integrates biochemical and structural data. We discuss our results and the structural model in the context of a possible mechanism of the TOB insertase.


Assuntos
Proteínas de Membrana/metabolismo , Membranas Mitocondriais/metabolismo , Neurospora crassa/metabolismo , Proteínas de Membrana/química , Modelos Moleculares , Conformação Proteica
19.
J Cell Biol ; 199(1): 125-35, 2012 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-23007651

RESUMO

Chaperones mediate protein folding and prevent deleterious protein aggregation in the cell. However, little is known about the biogenesis of chaperones themselves. In this study, we report on the biogenesis of the yeast mitochondrial Hsp70 (mtHsp70) chaperone, which is essential for the functionality of mitochondria. We show in vivo and in organello that mtHsp70 rapidly folds after its import into mitochondria, with its ATPase domain and peptide-binding domain (PBD) adopting their structures independently of each other. Importantly, folding of the ATPase domain but not of the PBD was severely affected in the absence of the Hsp70 escort protein, Hep1. We reconstituted the folding of mtHsp70, demonstrating that Hep1 and ATP/ADP were required and sufficient for its de novo folding. Our data show that Hep1 bound to a folding intermediate of mtHsp70. Binding of an adenine nucleotide triggered release of Hep1 and folding of the intermediate into native mtHsp70. Thus, Hep1 acts as a specialized chaperone mediating the de novo folding of an Hsp70 chaperone.


Assuntos
Proteínas de Choque Térmico HSP70/biossíntese , Mitocôndrias/metabolismo , Proteínas Mitocondriais/biossíntese , Proteínas de Saccharomyces cerevisiae/biossíntese , Saccharomyces cerevisiae/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
20.
Mol Biol Cell ; 23(22): 4335-46, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22993211

RESUMO

The vast majority of mitochondrial proteins are synthesized in the cytosol and transported into the organelle in a largely, if not completely, unfolded state. The proper function of mitochondria thus depends on folding of several hundreds of proteins in the various subcompartments of the organelle. Whereas folding of proteins in the mitochondrial matrix is supported by members of several chaperone families, very little is known about folding of proteins in the intermembrane space (IMS). We targeted dihydrofolate reductase (DHFR) as a model substrate to the IMS of yeast mitochondria and analyzed its folding. DHFR can fold in this compartment, and its aggregation upon heat shock can be prevented in an ATP-dependent manner. Yme1, an AAA (ATPases associated with diverse cellular activities) protease of the IMS, prevented aggregation of DHFR. Analysis of protein aggregates in mitochondria lacking Yme1 revealed the presence of a number of proteins involved in the establishment of mitochondrial ultrastructure, lipid metabolism, protein import, and respiratory growth. These findings explain the pleiotropic effects of deletion of YME1 and suggest an important role for Yme1 as a folding assistant, in addition to its proteolytic function, in the protein homeostasis of mitochondria.


Assuntos
Proteases Dependentes de ATP/fisiologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/fisiologia , Dobramento de Proteína , Proteínas de Saccharomyces cerevisiae/fisiologia , Tetra-Hidrofolato Desidrogenase/metabolismo , Proteases Dependentes de ATP/genética , Animais , Camundongos , Proteínas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
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