RESUMO
beta-Barrel proteins constitute a distinct class of mitochondrial outer membrane proteins. For import into mitochondria, their precursor forms engage the TOM complex. They are then relayed to the TOB complex, which mediates their insertion into the outer membrane. We studied the structure-function relationships of the core component of the TOB complex, Tob55. Tob55 precursors with deletions in the N-terminal domain were not affected in their targeting to and insertion into the mitochondrial outer membrane. Replacement of wild-type Tob55 by these deletion variants resulted in reduced growth of cells, and mitochondria isolated from such cells were impaired in their capacity to import beta-barrel precursors. The purified N-terminal domain was able to bind beta-barrel precursors in a specific manner. Collectively, these results demonstrate that the N-terminal domain of Tob55 recognizes precursors of beta-barrel proteins. This recognition may contribute to the coupling of the translocation of beta-barrel precursors across the TOM complex to their interaction with the TOB complex.
Assuntos
Proteínas Mitocondriais/biossíntese , Receptores de Superfície Celular/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Mutantes/metabolismo , Fenótipo , Porinas/metabolismo , Ligação Proteica , Dobramento de Proteína , Precursores de Proteínas/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/biossíntese , Deleção de Sequência , Relação Estrutura-AtividadeRESUMO
The outer membranes of mitochondria and chloroplasts are distinguished by the presence of beta-barrel membrane proteins. The outer membrane of Gram-negative bacteria also harbours beta-barrel proteins. In mitochondria these proteins fulfil a variety of functions such as transport of small molecules (porin/VDAC), translocation of proteins (Tom40) and regulation of mitochondrial morphology (Mdm10). These proteins are encoded by the nucleus, synthesized in the cytosol, targeted to mitochondria as chaperone-bound species, recognized by the translocase of the outer membrane, and then inserted into the outer membrane where they assemble into functional oligomers. Whereas some knowledge has been accumulated on the pathways of insertion of proteins that span cellular membranes with alpha-helical segments, very little is known about how beta-barrel proteins are integrated into lipid bilayers and assembled into oligomeric structures. Here we describe a protein complex that is essential for the topogenesis of mitochondrial outer membrane beta-barrel proteins (TOB). We present evidence that important elements of the topogenesis of beta-barrel membrane proteins have been conserved during the evolution of mitochondria from endosymbiotic bacterial ancestors.
Assuntos
Evolução Molecular , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Proteínas de Membrana/biossíntese , Proteínas de Membrana/química , Neurospora crassa/metabolismo , Dicroísmo Circular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Bicamadas Lipídicas/metabolismo , Substâncias Macromoleculares , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Neurospora crassa/química , Neurospora crassa/citologia , Ligação Proteica , Estrutura Secundária de Proteína , Transporte Proteico , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Blue native gel electrophoresis (BNGE) is a powerful tool for analyzing native protein complexes from biological membranes as well as water-soluble proteins. It can be used for determining relative molecular masses of protein complexes and their subunit composition and for the detection of subcomplexes. We describe the analysis by BNGE of in vitro import reactions composed of radiolabeled precursor proteins and isolated mitochondria. Such an analysis is a powerful tool to follow import intermediates and to study assembly of protein complexes. Analysis of import reactions by BNGE provides information on the molecular mass of the complex with which the imported precursor is associated. In addition, components of such a complex can be identified by incubating the mitochondrial lysate with either soluble antibodies or antibodies coupled to protein A matrix. The binding of soluble antibodies to specific complexes results in an observed shift in their apparent molecular mass (antibody shift). Alternatively, addition of matrix-bound antibodies followed by removal of the matrix from the mixture will result in depletion of the specific complex from the mitochondrial lysate (antibody depletion). The experimental details of these techniques are described.
Assuntos
Eletroforese/métodos , Proteínas Mitocondriais/metabolismo , Acrilamida , Anticorpos , Marcação por Isótopo , Membranas Artificiais , Polivinil , Precursores de Proteínas/metabolismo , Transporte Proteico , Saccharomyces cerevisiae/metabolismo , SolubilidadeRESUMO
Cell-permeable peptides were evaluated for a quantitatively controlled import of small molecules. The dependence of the import efficiency on the fluorophore, on the position of the fluorophore as well as on the nature of the cargo were addressed. Cellular uptake was quantitated by flow cytometry and fluorescence correlation microscopy (FCM). Fluorophores with different spectral characteristics, covering the whole visible spectral range, were selected in order to enable the simultaneous detection of several cell-permeable peptide constructs. The transcytosis sequences were based either on the sequence of the Antennapedia homeodomain protein (AntpHD)-derived penetratin peptide or the Kaposi fibroblast growth factor (FGF)-derived membrane translocating sequence (MTS)-peptide. In general, the AntpHD-derived peptides had a three- to fourfold higher import efficiency than the MTS-derived peptides. In spite of the very different physicochemical characteristics of the fluorophores, the import efficiencies for analogues labelled at different positions within the sequence of the import peptides showed a strong positive correlation. However, even for peptide cargos of very similar size, pronounced differences in import efficiency were observed. The use of cell-permeable peptide/cargo constructs for intracellular analyses of structure-function relationships therefore requires the determination of the intracellular concentrations for each construct individually.
Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Proteínas Nucleares , Peptídeos/metabolismo , Fatores de Transcrição , Sequência de Aminoácidos , Proteína do Homeodomínio de Antennapedia , Proteínas de Transporte/metabolismo , Permeabilidade da Membrana Celular , Peptídeos Penetradores de Células , Citometria de Fluxo , Fluoresceínas , Corantes Fluorescentes , Proteínas de Homeodomínio/metabolismo , Humanos , Microscopia Confocal , Rodaminas , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
The translocase of the outer mitochondrial membrane (TOM) complex is the general entry site into the organelle for newly synthesized proteins. Despite its central role in the biogenesis of mitochondria, the assembly process of this complex is not completely understood. Mim1 (mitochondrial import protein 1) is a mitochondrial outer membrane protein with an undefined role in the assembly of the TOM complex. The protein is composed of an N-terminal cytosolic domain, a central putative transmembrane segment (TMS) and a C-terminal domain facing the intermembrane space. Here we show that Mim1 is required for the integration of the import receptor Tom20 into the outer membrane. We further investigated what the structural characteristics allowing Mim1 to fulfil its function are. The N- and C-terminal domains of Mim1 are crucial neither for the function of the protein nor for its biogenesis. Thus, the TMS of Mim1 is the minimal functional domain of the protein. We show that Mim1 forms homo-oligomeric structures via its TMS, which contains two helix-dimerization GXXXG motifs. Mim1 with mutated GXXXG motifs did not form oligomeric structures and was inactive. With all these data taken together, we propose that the homo-oligomerization of Mim1 allows it to fulfil its function in promoting the integration of Tom20 into the mitochondrial outer membrane.
Assuntos
Proteínas de Membrana/metabolismo , Membranas Mitocondriais/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Dimerização , Proteínas de Membrana/química , Proteínas de Transporte da Membrana Mitocondrial , Membranas Mitocondriais/química , Neurospora crassa/química , Neurospora crassa/genética , Neurospora crassa/metabolismo , Estrutura Terciária de Proteína , Receptores Citoplasmáticos e Nucleares/química , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Schizosaccharomyces/química , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismoRESUMO
The mitochondrial outer membrane mediates numerous interactions between the metabolic and genetic systems of mitochondria and the rest of the eukaryotic cell. We performed a proteomic study to discover novel functions of components of the mitochondrial outer membrane. Proteins of highly pure outer membrane vesicles (OMV) from Neurospora crassa were identified by a combination of LC-MS/MS of tryptic peptide digests and gel electrophoresis of solubilized OMV proteins, followed by their identification using MALDI-MS PMF. Among the 30 proteins found in at least three of four separate analyses were 23 proteins with known functions in the outer membrane. These included components of the import machinery (the TOM and TOB complexes), a pore-forming component (porin), and proteins that control fusion and fission of the organelle. In addition, proteins playing a role in various biosynthetic pathways, whose intracellular location had not been established previously, could be localized to the mitochondrial outer membrane. Thus, the proteome of the outer membrane can help in identifying new mitochondria-related functions.
Assuntos
Proteínas Fúngicas/química , Membranas Intracelulares/química , Mitocôndrias/química , Neurospora crassa/química , Proteoma , DNA Recombinante , Eletroforese em Gel de Poliacrilamida , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Signal-anchored proteins are a class of mitochondrial outer membrane proteins that expose a hydrophilic domain to the cytosol and are anchored to the membrane by a single transmembrane domain in the N-terminal region. Like the vast majority of mitochondrial proteins, signal-anchored proteins are synthesized on cytosolic ribosomes and are subsequently imported into the organelle. We have studied the mechanisms by which precursors of these proteins are recognized by the mitochondria and are inserted into the outer membrane. The import of signal-anchored proteins was found to be independent of the known import receptors, Tom20 and Tom70, but to require the major Tom component, Tom40. In contrast to precursors destined to internal compartments of mitochondria and those of outer membrane beta-barrel proteins, precursors of signal-anchored proteins appear not to be inserted via the general import pore. Taken together, we propose a novel pathway for insertion of these proteins into the outer membrane of mitochondria.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana Transportadoras/química , Proteínas de Transporte da Membrana Mitocondrial/análise , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Modelos Moleculares , Transporte Proteico , Receptores Citoplasmáticos e Nucleares/química , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Transdução de SinaisRESUMO
All mitochondrial precursor proteins studied so far are recognized initially at the surface of the organelle by the translocase of the outer membrane (TOM complex). Precursors of beta-barrel proteins are transferred further to another complex in the outer membrane that mediates their topogenesis (TOB complex). Tob55 is an essential component of the TOB complex in that it constitutes the core element of the protein-conducting pore. The other two components of the TOB complex are Tob38, which builds a functional TOB core complex with Tob55, and Mas37, a peripheral member of the complex. We have investigated the biogenesis of the TOB complex. Reduced insertion of the Tob55 precursor in the absence of Tom20 and Tom70 argues for initial recognition of the precursor of Tob55 by the import receptors. Next, it is transferred through the import channel formed by Tom40. Variants of the latter protein influenced the insertion of Tob55. Assembly of newly synthesized Tob55 into preexisting TOB complexes, as analyzed by blue native gel electrophoresis, depended on Tob38 but did not require Mas37. Surprisingly, both the association of Mas37 precursor with mitochondria and its assembly into the TOB complex were not affected by mutation in the TOM complex. Mas37 assembled directly with the TOB core complex. Hence, the biogenesis of Mas37 represents a novel import pathway of mitochondrial proteins.
Assuntos
Proteínas Mitocondriais/biossíntese , Proteínas de Saccharomyces cerevisiae/biossíntese , Transporte Biológico , Substâncias Macromoleculares , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Precursores de Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The translocase of the outer mitochondrial membrane (TOM complex) is the general entry site for newly synthesized proteins into mitochondria. This complex is essential for the formation and maintenance of mitochondria. Here, we report on the role of the integral outer membrane protein, Mim1 (mitochondrial import), in the biogenesis of mitochondria. Depletion of Mim1 abrogates assembly of the TOM complex and results in accumulation of Tom40, the principal constituent of the TOM complex, as a low-molecular-mass species. Like all mitochondrial beta-barrel proteins, the precursor of Tom40 is inserted into the outer membrane by the TOB complex. Mim1 is likely to be required for a step after this TOB-complex-mediated insertion. Mim1 is a constituent of neither the TOM complex nor the TOB complex; rather, it seems to be a subunit of another, as yet unidentified, complex. We conclude that Mim1 has a vital and specific function in the assembly of the TOM complex.
Assuntos
Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/química , Mitocôndrias/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Membranas Intracelulares/química , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de SequênciaRESUMO
A protocol was developed for performing intracellular concentration measurements in flat adherent tissue culture cells by fluorescence correlation microscopy (FCM). Determination of the number of molecules in the confocal detection volume had to account for background fluorescence caused by molecules adsorbed to the surface of the measurement chamber. Such a background signal leads to a decrease in the amplitude of the autocorrelation function, and thereby to the calculation of an erroneously high number of molecules. Because of the spatial heterogeneity of the background intensity, a method was devised by which its contribution to the total fluorescence could be determined directly from each individual autocorrelation measurement. This method was applied to a comparison of the import efficiencies of different cell-permeable peptides at nanomolar concentrations. The Antennapedia homeodomain-derived peptide penetratin was imported about three times as efficient as the basic fibroblast growth factor-derived MTS peptide. Both peptides equilibrated between cytoplasm and nucleus. A relatively high mobility of these molecules inside the cells indicated that they may be rapidly degraded by cytosolic proteases. Based on these results, it will be possible to determine intracellular concentrations of inhibitors linked to import peptides directly by FCM at nanomolar concentrations and to optimise such constructs for proteolytic stability.
Assuntos
Proteínas de Transporte/análise , Fator 2 de Crescimento de Fibroblastos/análise , Proteínas de Transporte/farmacocinética , Permeabilidade da Membrana Celular , Peptídeos Penetradores de Células , Fator 2 de Crescimento de Fibroblastos/farmacocinética , Corantes Fluorescentes , Melanoma , Microscopia de Fluorescência , Coloração e Rotulagem , Células Tumorais CultivadasRESUMO
We have studied the topogenesis of a class of mitochondrial outer membrane proteins that expose a hydrophilic domain to the cytosol and are anchored to the membrane by a single transmembrane domain in the N-terminal region. To determine the role of these latter sequences in the targeting and insertion of such proteins we took two approaches. First, a functional complementation assay was used to define the structural elements that together with the anchor domain make up the topogenic signal. Moderate hydrophobicity of the transmembrane domain was found to be the most important requirement. Variants with a scrambled sequence of the membrane-spanning segment were only partially functional suggesting that specificity in the amino acid sequence is also of considerable importance. A net positive charge at both flanking regions of the transmembrane domain contributes to the efficiency of targeting and membrane integration but is not an essential structural feature of this signal. Second, chimeras of Tom20, Tom70, and OM45 were generated that contained the cytosolic domain of Tom20 or Tom70 and the anchor domain of one of the other members of the class. These hybrid proteins were able to rescue the growth of cells lacking Tom20 or Tom70. Thus, anchor domains of outer membrane proteins are functionally interchangeable. They play only a minor role in the specific function of these proteins, but have a decisive role in topogenic signaling.
Assuntos
Proteínas Fúngicas/química , Proteínas de Membrana/química , Proteínas Mitocondriais/química , Sinais Direcionadores de Proteínas , Sequência de Aminoácidos , Interações Hidrofóbicas e Hidrofílicas , Membranas Intracelulares/química , Mitocôndrias/ultraestrutura , Estrutura Terciária de Proteína , Proteínas Recombinantes de FusãoRESUMO
Insertion of beta-barrel proteins into the outer membrane of mitochondria is mediated by the TOB complex. Known constituents of this complex are Tob55 and Mas37. We identified a novel component, Tob38. It is essential for viability of yeast and the function of the TOB complex. Tob38 is exposed on the surface of the mitochondrial outer membrane. It interacts with Mas37 and Tob55 and is associated with Tob55 even in the absence of Mas37. The Tob38-Tob55 core complex binds precursors of beta-barrel proteins and facilitates their insertion into the outer membrane. Depletion of Tob38 results in strongly reduced levels of Tob55 and Mas37 and the residual proteins no longer form a complex. Tob38-depleted mitochondria are deficient in the import of beta-barrel precursor proteins, but not of other outer membrane proteins or proteins of other mitochondrial subcompartments. We conclude that Tob38 has a crucial function in the biogenesis of beta-barrel proteins of mitochondria.