RESUMO
Blue native electrophoresis is one of the most popular techniques for mass estimation of native membrane proteins, but the use of non-optimal mass markers and acrylamide gels can compromise accuracy and reliability of the results. We present short protocols taking 10-30 min to prepare optimal sets of membrane protein markers from chicken, rat, mouse, and bovine heart. Especially heart materials from local supermarkets or butcher's shops, e.g. chicken or bovine heart, are ideal sources of high mass membrane protein standards. Considerable discrepancies between the migration behavior of membrane and soluble markers suggest using membrane protein markers for mass estimation of membrane proteins. Soluble standard proteins can be used, with some limitations, when soluble proteins are the focus. Principles and general rules for the determination of mass and oligomeric state of native membrane and soluble proteins are elaborated, and potential pitfalls are discussed.
Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Proteínas de Membrana/química , Animais , Bovinos , Galinhas , Camundongos , Peso Molecular , Miocárdio/química , RatosRESUMO
Here we study ATP synthase from human rho0 (rho zero) cells by clear native electrophoresis (CNE or CN-PAGE) and show that ATP synthase is almost fully assembled in spite of the absence of subunits a and A6L. This identifies subunits a and A6L as two of the last subunits to complete the ATP synthase assembly. Minor amounts of dimeric and even tetrameric forms of the large assembly intermediate were preserved under the conditions of CNE, suggesting that it associated further into higher order structures in the mitochondrial membrane. This result was reminiscent to the reduced amounts of dimeric and tetrameric ATP synthase from yeast null mutants of subunits e and g detected by CNE. The dimer/oligomer-stabilizing effects of subunits e/g and a/A6L seem additive in human and yeast cells. The mature IF1 inhibitor was specifically bound to the dimeric/oligomeric forms of ATP synthase and not to the monomer. Conversely, nonprocessed pre-IF1 still containing the mitochondrial targeting sequence was selectively bound to the monomeric assembly intermediate in rho0 cells and not to the dimeric form. This supports previous suggestions that IF1 plays an important role in the dimerization/oligomerization of mammalian ATP synthase and in the regulation of mitochondrial structure and function.
Assuntos
ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA/genética , DNA Mitocondrial/genética , Dimerização , Humanos , Técnicas In Vitro , ATPases Mitocondriais Próton-Translocadoras/genética , Dados de Sequência Molecular , Multimerização Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em TandemRESUMO
Replication of human cytomegalovirus (CMV) requires the expression of the viral mitochondria-localized inhibitor of apoptosis (vMIA). vMIA inhibits apoptosis by recruiting Bax to mitochondria, resulting in its neutralization. We show that vMIA decreases cell size, reduces actin polymerization, and induces cell rounding. As compared with vMIA-expressing CMV, vMIA-deficient CMV, which replicates in fibroblasts expressing the adenoviral apoptosis suppressor E1B19K, induces less cytopathic effects. These vMIA effects can be separated from its cell death-inhibitory function because vMIA modulates cellular morphology in Bax-deficient cells. Expression of vMIA coincided with a reduction in the cellular adenosine triphosphate (ATP) level. vMIA selectively inhibited one component of the ATP synthasome, namely, the mitochondrial phosphate carrier. Exposure of cells to inhibitors of oxidative phosphorylation produced similar effects, such as an ATP level reduced by 30%, smaller cell size, and deficient actin polymerization. Similarly, knockdown of the phosphate carrier reduced cell size. Our data suggest that the cytopathic effect of CMV can be explained by vMIA effects on mitochondrial bioenergetics.
Assuntos
Apoptose , Infecções por Citomegalovirus/metabolismo , Citomegalovirus/fisiologia , Proteínas Imediatamente Precoces/fisiologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Virais/fisiologia , Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Citomegalovirus/genética , Efeito Citopatogênico Viral , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Fibroblastos/virologia , Células HeLa , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/toxicidade , Camundongos , Proteínas Mitocondriais/genética , Células NIH 3T3 , Fosforilação Oxidativa/efeitos dos fármacos , Polímeros/metabolismo , Proteínas Virais/genética , Proteínas Virais/toxicidade , Proteína X Associada a bcl-2/antagonistas & inibidores , Proteína X Associada a bcl-2/genéticaRESUMO
Here, we expand the application of blue native electrophoresis to the separation of mega protein complexes larger than 10 MDa by introducing novel large pore acrylamide gels. We tailored the bis-acrylamide cross-linker amounts relative to the acrylamide monomer to enlarge the pore size of acrylamide gels and to obtain elastic and sufficiently stable gels. The novel gel types were then used to search for suprastructures of mitochondrial respiratory supercomplexes, the hypothetical respiratory strings, or patches. We identified 4-8 MDa assemblies that contain respiratory complexes I, III, and IV and most likely represent dimers, trimers, and tetramers of respiratory supercomplexes. We also isolated multimeric respiratory supercomplexes with apparent masses of 35-45 MDa, the presumed core pieces of respiratory strings or patches. Electron microscopic investigations will be required to clarify whether the isolated assemblies of complexes are ordered and specific, as predicted for respiratory strings and patches in the mitochondrial membrane.
Assuntos
Proteoma/isolamento & purificação , Proteômica/métodos , Cor , Eletroforese , Mitocôndrias/química , Peso Molecular , PorosidadeRESUMO
A large repertoire of immunological methods permits monitoring the interaction of antibodies with their specific antigen. However, recognition of a protein by a conformation-specific antibody represents a challenge because native conditions must be kept throughout the assay. Native immunoblotting of blue native gels conserves the native state by using Tween 20 instead of methanol for the obligatory destaining of the blot membrane. We validate the new technique with a set of monoclonal antibodies against respiratory NADH:ubiquinone oxidoreductase.
Assuntos
Anticorpos Monoclonais/análise , Corantes/química , Complexo I de Transporte de Elétrons/análise , Eletroforese em Gel de Poliacrilamida/métodos , Immunoblotting/métodos , Corantes de Rosanilina/química , Yarrowia/química , Anticorpos Monoclonais/imunologia , Complexo I de Transporte de Elétrons/imunologiaRESUMO
We have developed an experimental approach that combines two powerful methods for proteomic analysis of large membrane protein complexes: blue native electrophoresis (BNE or BN-PAGE) and laser-induced liquid bead ion desorption (LILBID) MS. Protein complexes were separated by BNE and eluted from the gel. The masses of the constituents of the multiprotein complexes were obtained by LILBID MS, a detergent-tolerant method that is especially suitable for the characterisation of membrane proteins. High sensitivity and small sample volumes required for LILBID MS resulted in low demands on sample quantity. Eluate from a single band allowed assessing the mass of an entire multiprotein complex and its subunits. The method was validated with mitochondrial NADH:ubiquinone reductase from Yarrowia lipolytica. For this complex of 947 kDa, typically 30 microg or 32 pmol were sufficient to obtain spectra from which the subunit composition could be analysed. The resolution of this electrophoretic small-scale approach to the purification of native complexes was improved markedly by further separation on a second dimension of BNE. Starting from a subcellular fraction obtained by differential centrifugation, this allowed the purification and analysis of the constituents of a large multiprotein complex in a single LILBID spectrum.
Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Complexos Multiproteicos/química , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Proteínas de Bactérias/química , Lasers , Proteínas de Membrana/química , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Yarrowia/químicaRESUMO
Mitochondrial ATP synthase is mostly isolated in monomeric form, but in the inner mitochondrial membrane it seems to dimerize and to form higher oligomeric structures from dimeric building blocks. Following a period of electron microscopic single particle analyses that revealed an angular orientation of the membrane parts of monomeric ATP synthases in the dimeric structures, and after extensive studies of the monomer-monomer interface, the focus now shifts to the potentially dynamic state of the oligomeric structures, their potential involvement in metabolic regulation of mitochondria and cells, and to newly identified interactions like physical associations of complexes IV and V. Similarly, larger structures like respiratory strings that have been postulated to form from individual respiratory complexes and their supercomplexes, the respirasomes, come into the focus. Progress by structural investigations is paralleled by insights into the functional roles of respirasomes including substrate channelling and stabilization of individual complexes. Cardiolipin was found to be important for the structural stability of respirasomes which in turn is required to maintain cells and tissues in a healthy state. Defects in cardiolipin remodeling cause devastating diseases like Barth syndrome. Novel species-specific roles of respirasomes for the stability of respiratory complexes have been identified, and potential additional roles may be deduced from newly observed interactions of respirasomes with components of the protein import machinery and with the ADP/ATP translocator.
Assuntos
Transporte de Elétrons , Membranas Mitocondriais/química , ATPases Mitocondriais Próton-Translocadoras/química , Animais , Cardiolipinas/metabolismo , Bovinos , Dimerização , Membranas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Estrutura Quaternária de Proteína , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
ABC transporters represent one of the largest families of membrane proteins that are found in all three phyla of life. Mitochondria comprise up to four ABC systems, ABCB7/ATM1, ABCB10/MDL1, ABCB8 and ABCB6. These half-transporters, which assemble into homodimeric complexes, are involved in a number of key cellular processes, e.g. biogenesis of cytosolic iron-sulfur clusters, heme biosynthesis, iron homeostasis, multidrug resistance, and protection against oxidative stress. Here, we summarize recent advances and emerging themes in our understanding of how these ABC systems in the inner and outer mitochondrial membrane fulfill their functions in important (patho) physiological processes, including neurodegenerative and hematological disorders.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Trifosfato de Adenosina/metabolismo , Animais , Heme/biossíntese , Homeostase , Humanos , Hidrólise , Ferro/metabolismo , Proteínas Mitocondriais/genética , Modelos Moleculares , Filogenia , Processamento de Proteína Pós-TraducionalRESUMO
Specific protein-lipid interactions have been identified in X-ray structures of membrane proteins. The role of specifically bound lipid molecules in protein function remains elusive. In the current study, we investigated how phospholipids influence catalytic, spectral and electrochemical properties of the yeast respiratory cytochrome bc(1) complex and how disruption of a specific cardiolipin binding site in cytochrome c(1) alters respiratory supercomplex formation in mitochondrial membranes. Purified yeast cytochrome bc(1) complex was treated with phospholipase A(2). The lipid-depleted enzyme was stable but nearly catalytically inactive. The absorption maxima of the reduced b-hemes were blue-shifted. The midpoint potentials of the b-hemes of the delipidated complex were shifted from -52 to -82 mV (heme b(L)) and from +113 to -2 mV (heme b(H)). These alterations could be reversed by reconstitution of the delipidated enzyme with a mixture of asolectin and cardiolipin, whereas addition of the single components could not reverse the alterations. We further analyzed the role of a specific cardiolipin binding site (CL(i)) in supercomplex formation by site-directed mutagenesis and BN-PAGE. The results suggested that cardiolipin stabilizes respiratory supercomplex formation by neutralizing the charges of lysine residues in the vicinity of the presumed interaction domain between cytochrome bc(1) complex and cytochrome c oxidase. Overall, the study supports the idea, that enzyme-bound phospholipids can play an important role in the regulation of protein function and protein-protein interaction.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons/química , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Fosfolipídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Cardiolipinas/metabolismo , Domínio Catalítico/genética , Eletroquímica , Complexo III da Cadeia de Transporte de Elétrons/genética , Estabilidade Enzimática , Heme/química , Mitocôndrias/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oxirredução , Fosfolipases A2 , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , EspectrofotometriaRESUMO
Mass spectrometry of membrane protein complexes is still a methodological challenge due to hydrophobic and hydrophilic parts of the species and the fact that all subunits are bound non-covalently together. The present study with the novel laser induced liquid bead ion desorption mass spectrometry (LILBID-MS) reports on the determination of the subunit composition of the F(1)F(o)-ATP synthase from Bacillus pseudofirmus OF4, that of both bovine heart and, for the first time, of human heart mitochondrial F(1)F(o)-ATP synthases. Under selected buffer conditions the mass of the intact F(1)F(o)-ATP synthase of B. pseudofirmus OF4 could be measured, allowing the analysis of complex subunit stoichiometry. The agreement with theoretical masses derived from sequence databases is very good. A comparison of the ATP synthase subunit composition of 5 different ATPases reveals differences in the complexity of eukaryotic and bacterial ATP synthases. However, whereas the overall construction of eukaryotic enzymes is more complex than the bacterial ones, functionally important subunits are conserved among all ATPases.
Assuntos
ATPases Translocadoras de Prótons/química , Animais , Bacillus/enzimologia , Bovinos , Humanos , Espectrometria de Massas , Mitocôndrias/enzimologia , Subunidades Proteicas/químicaRESUMO
We disassembled monomeric and dimeric yeast ATP synthase under mild conditions to identify labile proteins and transiently stable subcomplexes that had not been observed before. Specific removal of subunits alpha, beta, oligomycin sensitivity conferring protein (OSCP), and h disrupted the ATP synthase at the gamma-alpha(3)beta(3) rotor-stator interface. Loss of two F(1)-parts from dimeric ATP synthase led to the isolation of a dimeric subcomplex containing membrane and peripheral stalk proteins thus identifying the membrane/peripheral stalk sectors immediately as the dimerizing parts of ATP synthase. Almost all subunit a was found associated with a ring of 10 c-subunits in two-dimensional blue native/SDS gels. We therefore postulate that c10a1-complex is a stable structure in resting ATP synthase until the entry of protons induces a breaking of interactions and stepwise rotation of the c-ring relative to the a-subunit in the catalytic mechanism. Dimeric subunit a was identified in SDS gels in association with two c10-rings suggesting that a c10a2c10-complex may constitute an important part of the monomer-monomer interface in dimeric ATP synthase that seems to be further tightened by subunits b, i, e, g, and h. In contrast to the monomer-monomer interface, the interface between dimers in higher oligomeric structures remains largely unknown. However, we could show that the natural inhibitor protein Inh1 is not required for oligomerization.
Assuntos
ATPases Mitocondriais Próton-Translocadoras/química , Complexos Multienzimáticos/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Proteínas Adaptadoras de Transdução de Sinal , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Dimerização , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Complexos Multienzimáticos/genética , Mutação , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Proteínas/química , Proteínas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteína Inibidora de ATPaseRESUMO
Permanent protein-protein interactions are commonly identified by co-purification of two or more protein components using techniques like co-immunoprecipitation, tandem affinity purification and native electrophoresis. Here we focus on blue-native electrophoresis, clear-native electrophoresis, high-resolution clear-native electrophoresis and associated techniques to identify stable membrane protein complexes and detergent-labile physiological supercomplexes. Hints for dynamic protein-protein interactions can be obtained using two-hybrid techniques but not from native electrophoresis and other protein isolation techniques except after covalent cross-linking of interacting proteins in vivo prior to protein separation.
Assuntos
Eletroforese em Gel Bidimensional/métodos , Mapeamento de Interação de Proteínas/métodos , Proteínas/análise , Animais , Eletroforese em Gel Bidimensional/tendências , Humanos , Mapeamento de Interação de Proteínas/tendências , Proteínas/metabolismoRESUMO
Mitochondria of the strictly aerobic yeast Yarrowia lipolytica contain respiratory complex I with close functional and structural similarity to the mammalian enzyme. Unlike mammalian mitochondria, however, Yarrowia mitochondria have been thought not to contain supercomplexes. Here, we identify respiratory supercomplexes composed of complexes I, III and IV also in Y. lipolytica. Evidence for dimeric complex I suggests further association of respiratory supercomplexes into respiratory strings or patches. Similar supercomplex organization in Yarrowia and mammalian mitochondria further makes this aerobic yeast a useful model for the human oxidative phosphorylation system. The analysis of supercomplexes and their constituent complexes was made possible by 2-D native electrophoresis, i.e. by using native electrophoresis for both dimensions. Digitonin and blue-native electrophoresis were generally applied for the initial separation of supercomplexes followed by less mild native electrophoresis variants in the second dimension to release the individual complexes from the supercomplexes. Such 2-D native systems are useful means to identify the constituent proteins and their copy numbers in detergent-labile physiological assemblies, since they can reduce the complexity of supramolecular systems to the level of individual complexes.
Assuntos
Proteínas Mitocondriais/química , Complexos Multienzimáticos/química , Bombas de Próton/química , Yarrowia/química , Animais , Proteínas de Bactérias/química , Bovinos , Eletroforese em Gel Bidimensional , Mitocôndrias/química , Mitocôndrias Cardíacas/química , Modelos Biológicos , Modelos Moleculares , Subunidades Proteicas/químicaRESUMO
Specific modules and subcomplexes like F(1) and F(0)-parts, F(1)-c subcomplexes, peripheral and central stalks, and the rotor part comprising a ring of c-subunits with attached subunits gamma, delta, and epsilon can be identified in yeast and mammalian ATP synthase. Four subunits, alpha(3)beta(3), OSCP, and h, seem to form a structural entity at the extramembranous rotor/stator interface (gamma/alpha(3)beta(3)) to hold and stabilize the rotor in the holo-enzyme. The intramembranous rotor/stator interface (c-ring/a-subunit) must be dynamic to guarantee unhindered rotation. Unexpectedly, a c(10)a-assembly could be isolated with almost quantitive yield suggesting that an intermediate step in the rotating mechanism was frozen under the conditions used. Isolation of dimeric a-subunit and (c(10))(2)a(2)-complex from dimeric ATP synthase suggested that the a-subunit stabilizes the same monomer-monomer interface that had been shown to involve also subunits e, g, b, i, and h. The natural inhibitor protein Inh1 does not favor oligomerization of yeast ATP synthase. Other candidates for the oligomerization of dimeric ATP synthase building blocks are discussed, e.g. the transporters for inorganic phosphate and ADP/ATP that had been identified as constituents of ATP synthasomes. Independent approaches are presented that support previous reports on the existence of ATP synthasomes in the mitochondrial membrane.
Assuntos
ATPases Mitocondriais Próton-Translocadoras/química , Sequência de Aminoácidos , Animais , Domínio Catalítico , Bovinos , Mamíferos , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Modelos Moleculares , Fragmentos de Peptídeos/química , Conformação Proteica , Processamento de Proteína Pós-Traducional , Subunidades Proteicas/química , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Classical 2-D electrophoresis (IEF/SDS 2-DE) using isoelectric focusing (IEF) and SDS-PAGE for the second dimension offers very high resolution for the separation of complex protein mixtures, but hydrophobic proteins can aggregate and are considerably under-represented in these 2-D gels. Non-classical 2-DE, as described here, summarizes several heterogeneous techniques, some of which, like BAC/SDS 2-DE and doubled SDS-polyacrylamide gel electrophoresis (dSDS-PAGE), intend to isolate the difficult hydrophobic proteins that are not accessible by classical 2-DE. Other types of non-classical 2-DE start with 1-D separation of native proteins and complexes, like blue-native electrophoresis (BNE), clear-native electrophoresis (CNE), and high-resolution clear-native electrophoresis (hrCNE). These electrophoretic techniques can substitute for chromatographic isolation of protein complexes, and can even isolate supramolecular physiological assemblies. Subsequent resolution in second dimension can be denaturing to resolve the subunits of complexes, as exemplified with BNE/SDS 2-DE, or native like in BNE/BNE 2-DE (the latter using different cathode buffers for 1-D BNE and 2-D BNE). After isolation of highly pure membrane protein complexes by two native electrophoretic separations, the separation protocol may be finished by denaturing 2-DE like BAC/SDS or doubled SDS-PAGE. Thus, a four-dimensional electrophoretic system with minimal loss of protein results that is useful as an efficient micro-scale protein separation protocol, e.g. for mass spectrometric analyses.
Assuntos
Eletroforese em Gel Bidimensional/métodos , Proteínas Mitocondriais/isolamento & purificação , Fragmentos de Peptídeos/análise , Proteômica/métodos , Animais , Bovinos , Eletroforese em Gel de Poliacrilamida , Mitocôndrias Cardíacas/metabolismo , RatosRESUMO
1-D native electrophoresis is used for the separation of individual proteins, protein complexes, and supercomplexes. Stable and labile protein-protein interactions can be identified depending on detergent and buffer conditions. 1-D native gels are immediately applicable for in-gel detection of fluorescent-labeled proteins and for in-gel catalytic activity assays. 1-D native gels and blots are used to determine native mass and oligomeric state of membrane proteins. Protein extracts from 1-D native gels are used for generation of antibodies, for proteomic work, and for advanced structural investigations. 2-D separation of subunits of protein complexes by SDS-PAGE is mostly used for immunological and proteomic studies. Following the discussion of these general features, specific applications of native electrophoresis techniques in various research fields are highlighted: immunological and receptor studies, biogenesis and assembly of membrane protein complexes, protein import into organelles, dynamics of proteasomes, proteome and subproteome investigations, the identification and quantification of mitochondrial alterations in apoptosis, carcinogenesis, and neurodegenerative disorders like Parkinson's disease, Alzheimer's disease, and the vast variety of mitochondrial encephalomyopathies.
Assuntos
Eletroforese em Gel Bidimensional/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Proteoma/análise , Proteômica/métodos , Animais , Eletroforese em Gel Bidimensional/tendências , Eletroforese em Gel de Poliacrilamida/tendências , Humanos , Encefalomiopatias Mitocondriais/metabolismo , Encefalomiopatias Mitocondriais/patologia , Proteínas Mitocondriais/análise , Ligação Proteica , Transporte Proteico , Proteoma/metabolismo , Proteômica/tendênciasRESUMO
Dimerization or oligomerization of ATP synthase has been proposed to play an important role for mitochondrial cristae formation and to be involved in regulating ATP synthase activity. We found comparable oligomycin-sensitive ATPase activity for monomeric and oligomeric ATP synthase suggesting that oligomerization/monomerization dynamics are not directly involved in regulating ATP synthase activity. Binding of the natural IF1 inhibitor protein has been shown to induce dimerization of F1-subcomplexes. This suggested that binding of IF1 might also dimerize holo ATP synthase, and possibly link dimerization and inhibition. Analyzing mitochondria of human rho zero cells that contain mitochondria but lack mitochondrial DNA, we identified three subcomplexes of ATP synthase: (i) F1 catalytic domain, (ii) F1-domain with bound IF1, and (iii) F1-c subcomplex with bound IF1 and a ring of subunits c. Since both IF1 containing subcomplexes were present in monomeric state and exhibited considerably reduced ATPase activity as compared to the third subcomplex lacking IF1, we postulate that inhibition and induction of dimerization of F1-subcomplexes by IF1 are independent events. F1-subcomplexes were also found in mitochondria of patients with specific mitochondrial disorders, and turned out to be useful for the clinical differentiation between various types of mitochondrial biosynthesis disorders. Supramolecular associations of respiratory complexes, the "respirasomes", seem not to be the largest assemblies in the structural organization of the respiratory chain, as suggested by differential solubilization of mitochondria and electron microscopic analyses of whole mitochondria. We present a model for a higher supramolecular association of respirasomes into a "respiratory string".
Assuntos
Mitocôndrias Cardíacas/metabolismo , Complexos Multienzimáticos/metabolismo , Fosforilação Oxidativa , Animais , Respiração Celular/fisiologia , Dimerização , Eletroforese em Gel Bidimensional , Humanos , Mitocôndrias Cardíacas/enzimologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Modelos Biológicos , RatosRESUMO
Respiratory chain complexes are fragments of larger structural and functional units, the respiratory chain supercomplexes or "respirasomes", which exist in bacterial and mitochondrial membranes. Supercomplexes of mitochondria and bacteria contain complexes III, IV, and complex I, with the notable exception of Saccharomyces cerevisiae, which does not possess complex I. These supercomplexes often are stable to sonication but sensitive to most detergents except digitonin. In S. cerevisiae, a major component linking complexes III and IV together is cardiolipin.In Paracoccus denitrificans, complex I itself is rather detergent-sensitive and thus could not be obtained in detergent-solubilized form so far. However, it can be isolated as part of a supercomplex. Stabilization of complex I by binding to complex III was also found in human mitochondria. Further functional roles of the organization in a supercomplex are catalytic enhancement by reducing diffusion distances of substrates or, depending on the organism, channelling of the substrates quinone and cytochrome c. This makes redox reactions less dependent of midpoint potentials of substrates, and permits electron flow at low degree of substrate reduction.A dimeric state of ATP synthase seems to be specific for mitochondria. Exclusively, monomeric ATP synthase was found in Acetobacterium woodii, in P. denitrificans, and in spinach chloroplasts.
Assuntos
Complexos de ATP Sintetase/química , Complexos de ATP Sintetase/isolamento & purificação , Animais , ATPases Bacterianas Próton-Translocadoras/química , Cardiolipinas/química , Bovinos , ATPases de Cloroplastos Translocadoras de Prótons/química , Dimerização , Complexo I de Transporte de Elétrons , Complexo III da Cadeia de Transporte de Elétrons/química , Humanos , Mitocôndrias Cardíacas/enzimologia , ATPases Mitocondriais Próton-Translocadoras/química , Modelos Moleculares , NADH NADPH Oxirredutases/química , Oxirredução , Fosforilação Oxidativa , Paracoccus denitrificans/enzimologia , Saccharomyces cerevisiae/enzimologiaRESUMO
Here we present a first assessment of the subunit inventory of mitochondrial complex I from the obligate aerobic yeast Yarrowia lipolytica. A total of 37 subunits were identified. In addition to the seven central, nuclear coded, and the seven mitochondrially coded subunits, 23 accessory subunits were found based on 2D electrophoretic and mass spectroscopic analysis in combination with sequence information from the Y. lipolytica genome. Nineteen of the 23 accessory subunits are clearly conserved between Y. lipolytica and mammals. The remaining four accessory subunits include NUWM, which has no apparent homologue in any other organism and is predicted to contain a single transmembrane domain bounded by highly charged extramembraneous domains. This structural organization is shared among a group of 7 subunits in the Y. lipolytica and 14 subunits in the mammalian enzyme. Because only five of these subunits display significant evolutionary conservation, their as yet unknown function is proposed to be structure- rather than sequence-specific. The NUWM subunit could be assigned to a hydrophobic subcomplex obtained by fragmentation and sucrose gradient centrifugation. Its position within the membrane arm was determined by electron microscopic single particle analysis of Y. lipolytica complex I decorated with a NUWM-specific monoclonal antibody.
Assuntos
Complexo I de Transporte de Elétrons/química , Mitocôndrias/enzimologia , Subunidades Proteicas/análise , Yarrowia/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Complexo I de Transporte de Elétrons/metabolismo , Eletroforese em Gel Bidimensional , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , Subunidades Proteicas/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Yarrowia/genéticaRESUMO
In bacteria, the Sec-protein transport complex facilitates the passage of most secretory and membrane proteins across and into the plasma membrane. The core complex SecYEG forms the protein channel and engages either ribosomes or the ATPase SecA, which drive translocation of unfolded polypeptide chains through the complex and into the periplasmic space. Escherichia coli SecYEG forms dimers in membranes, but in detergent solution the population of these dimers is low. However, we find that stable dimers can be assembled by the addition of a monoclonal antibody. Normally, a stable SecYEG-SecA complex can only form on isolated membranes or on reconstituted proteo-liposomes. The antibody-stabilised SecYEG dimer binds one SecA molecule in detergent solution. In the presence of AMPPNP, a non-hydrolysable analogue of ATP, a complex forms containing one antibody and two each of SecYEG and SecA. SecYEG monomers or tetramers do not associate to a significant degree with SecA. The observed variability in the stoichiometry of SecYEG and SecA association and its nucleotide modulation may be important and necessary for the protein translocation reaction.