RESUMO
Numerous components have been identified that participate at various stages in the biogenesis of mitochondria. For many of these components, their specific functions have recently been defined through detailed investigations of the molecular mechanisms underlying protein targeting, translocation across the mitochondrial outer and inner membranes, membrane insertion, suborganellar sorting, and protein folding.
Assuntos
Compartimento Celular , Mitocôndrias/metabolismo , Proteínas/metabolismo , Transporte Biológico , Membranas Intracelulares/metabolismo , Modelos Biológicos , Dobramento de Proteína , Precursores de Proteínas/metabolismoRESUMO
Mitochondria import the majority of their proteins from the cytosol. At the mitochondrial outer membrane, import is initiated through a series of reactions, which include preprotein recognition, unfolding, insertion and translocation. These processes are facilitated by a multisubunit complex, the TOM complex. Specific roles can now be assigned to several components of this complex. Although the import machinery of the outer membrane can insert and translocate a few proteins on its own, completion of translocation o f most preproteins is dependent upon coupling to both the membrane potential and mt-Hsp70/ATP-driven transport across the inner membrane, mediated by the TIM complex.
RESUMO
Import of nuclear-encoded mitochondrial preproteins is mediated by a general translocase in the outer membrane, the TOM complex, and by two distinct translocases in the mitochondrial inner membrane, the TIM23 complex and the TIM22 complex. Both TIM complexes cooperate with the TOM complex but facilitate import of different classes of precursor proteins. Precursors with an N-terminal presequence are imported via the TIM23 complex, whereas mitochondrial carrier proteins require the TIM22 complex for insertion into the inner membrane. This review discusses recent advances in understanding the structure and function of the translocases of the inner membrane and the possible role of Tim proteins in the development of the Mohr-Tranebjaerg syndrome, a mitochondrial disorder leading to neurodegeneration.
Assuntos
Proteínas de Transporte , Proteínas de Membrana , Proteínas de Membrana Transportadoras , Mitocôndrias/fisiologia , Proteínas de Saccharomyces cerevisiae , Animais , Transporte Biológico , Humanos , Proteínas de Transporte da Membrana Mitocondrial , Miopatias Mitocondriais , Proteínas do Complexo de Importação de Proteína Precursora MitocondrialRESUMO
Tom40 is an essential component of the preprotein translocase of the mitochondrial outer membrane (TOM complex) in which it constitutes the core element of the protein conducting pore. We have investigated the biogenesis of Tom40. Tom40 is inserted into the outer membrane by the TOM complex. Initially, Tom40 is bound as a monomer at the mitochondrial surface. The import receptor Tom20 is involved in this initial step; it stimulates both binding and efficient insertion of the Tom40 precursor. This step is followed by the formation of a further intermediate at which the Tom40 precursor is partially inserted into the outer membrane. Finally, Tom40 is integrated into preexisting TOM complexes. Efficient import appears to require the Tom40 precursor to be in a partially folded conformation. Neither the NH(2) nor the COOH termini are necessary to target Tom40 to the outer membrane. However, the NH(2)-terminal segment is required for Tom40 to become assembled into the TOM complex. A model for the biogenesis of Tom40 is presented.
Assuntos
Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Mitocôndrias/enzimologia , Complexos Multienzimáticos/metabolismo , Receptores Citoplasmáticos e Nucleares , Proteínas de Saccharomyces cerevisiae , Transporte Biológico , Reagentes de Ligações Cruzadas , Endopeptidases/metabolismo , Membranas Intracelulares/química , Cinética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial , Modelos Biológicos , Peso Molecular , Complexos Multienzimáticos/química , Neurospora crassa , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Precursores de Proteínas/química , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas/química , Sinais Direcionadores de Proteínas/genética , Sinais Direcionadores de Proteínas/metabolismo , Deleção de Sequência , TemperaturaRESUMO
Nuclear-encoded proteins destined for mitochondria must cross the outer or both outer and inner membranes to reach their final sub-mitochondrial locations. While the inner membrane can translocate preproteins by itself, it is not known whether the outer membrane also contains an endogenous protein translocation activity which can function independently of the inner membrane. To selectively study the protein transport into and across the outer membrane of Neurospora crassa mitochondria, outer membrane vesicles were isolated which were sealed, in a right-side-out orientation, and virtually free of inner membranes. The vesicles were functional in the insertion and assembly of various outer membrane proteins such as porin, MOM19, and MOM22. Like with intact mitochondria, import into isolated outer membranes was dependent on protease-sensitive surface receptors and led to correct folding and membrane integration. The vesicles were also capable of importing a peripheral component of the inner membrane, cytochrome c heme lyase (CCHL), in a receptor-dependent fashion. Thus, the protein translocation machinery of the outer mitochondrial membrane can function as an independent entity which recognizes, inserts, and translocates mitochondrial preproteins of the outer membrane and the intermembrane space. In contrast, proteins which have to be translocated into or across the inner membrane were only specifically bound to the vesicles, but not imported. This suggests that transport of such proteins involves the participation of components of the intermembrane space and/or the inner membrane, and that in these cases the outer membrane translocation machinery has to act in concert with that of the inner membrane.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Mitocôndrias/metabolismo , Precursores de Proteínas/metabolismo , Receptores Citoplasmáticos e Nucleares , Transporte Biológico , Fracionamento Celular , Proteínas Fúngicas/metabolismo , Membranas Intracelulares/metabolismo , Neurospora crassa , Receptores de Superfície Celular/metabolismoRESUMO
Purified mitochondrial ribosomes (60S) have been isolated from locust flight muscle. Purification could be achieved after lysis of mitochondria in 0.055 M MgCl(2). Mitochondrial 60S and cytoplasmic 80S ribosomes were investigated by electron microscopy in tissue sections, in sections of pellets of isolated ribosomes, and by negative staining of ribosomal suspensions. In negatively stained preparations, mitochondrial ribosomes show dimensions of approximately 270 x 210 x 215 A; cytoplasmic ribosomes measure approximately 295 x 245 x 255 A. From these values a volume ratio of mitochondrial to cytoplasmic ribosomes of 1: 1.5 was estimated. Despite their different sedimentation constants, mitochondrial ribosomes after negative staining show a morphology similar to that of cytoplasmic ribosomes. Both types of particles show bipartite profiles which are interpreted as "frontal views" and "lateral views." In contrast to measurements on negatively stained particles, the diameter of mitochondrial ribosomes in tissue sections is approximately 130 A, while the diameter of cytoplasmic ribosomes is approximately 180-200 A. These data suggest a volume ratio of mitochondrial to cytoplasmic ribosomes of 1:3. Subunits of mitochondrial ribosomes (40S and 25S) were obtained by incubation under dissociating conditions before fixation in glutaraldehyde. After negative staining, mitochondrial large (40S) subunits show rounded profiles with a shallow groove on a flattened side of the profile. Mitochondrial small subunits (25S) display elongated, triangular profiles.
Assuntos
Citoplasma , Gafanhotos/anatomia & histologia , Mitocôndrias Musculares , Ribossomos , Animais , Centrifugação com Gradiente de Concentração , Microscopia Eletrônica , Coloração e RotulagemRESUMO
Import of proteins into the mitochondrial matrix requires translocation across two membranes. Translocational intermediates of mitochondrial proteins, which span the outer and inner membrane simultaneously and thus suggest that translocation occurs in one step, have recently been described (Schleyer, M., and W. Neupert, 1985, Cell, 43:339-350). In this study we present evidence that distinct membrane areas are involved in the translocation process. Mitochondria that had lost most of their outer membrane by digitonin treatment (mitoplasts) still had the ability to import proteins. Import depended on proteinaceous structures of the residual outer membrane and on a factor that is located between the outer and inner membranes and that could be extracted with detergent plus salt. Translocational intermediates, which had been preformed before fractionation, remained with the mitoplasts under conditions where most of the outer membrane was subsequently removed. Submitochondrial vesicles were isolated in which translocational intermediates were enriched. Immunocytochemical studies also suggested that the translocational intermediates are located in areas where outer and inner membranes are in close proximity. We conclude that the membrane-potential-dependent import of precursor proteins involves translocation contact sites where the two membranes are closely apposed and are linked in a stable manner.
Assuntos
Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Transporte Biológico , Digitonina/farmacologia , Proteínas Fúngicas/metabolismo , Membranas Intracelulares/ultraestrutura , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Neurospora crassa/metabolismo , Neurospora crassa/ultraestrutura , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Hsp78, a member of the family of Clp/Hsp100 proteins, exerts chaperone functions in mitochondria of S. cerevisiae which overlap with those of mitochondrial Hsp70. In the present study, the role of Hsp78 under extreme stress was analyzed. Whereas deletion of HSP78 does not affect cell growth at temperatures up to 39 decrees C and cellular thermotolerance at 50 degrees C, Hsp78 is crucial for maintenance of respiratory competence and for mitochondrial genome integrity under severe temperature stress (mitochondrial thermotolerance). Mitochondrial protein synthesis is identified as a thermosensitive process. Reactivation of mitochondrial protein synthesis after heat stress depends on the presence of Hsp78, though Hsp78 does not confer protection against heat-inactivation to this process. Hsp78 appears to act in concert with other mitochondrial chaperone proteins since a conditioning pretreatment of the cells to induce the cellular heat shock response is required to maintain mitochondrial functions under severe temperature stress. When expressed in the cytosol, Hsp78 can substitute for the homologous heat shock protein Hsp104 in mediating cellular thermotolerance, suggesting a conserved mode of action of the two proteins. Thus, proteins of the Clp/Hsp100-family located in the cytosol and within mitochondria confer compartment-specific protection against heat damage to the cell.
Assuntos
Compartimento Celular/fisiologia , Proteínas Fúngicas/fisiologia , Proteínas de Choque Térmico/fisiologia , Mitocôndrias/fisiologia , Proteínas de Saccharomyces cerevisiae , Sobrevivência Celular/fisiologia , Citosol/química , Proteínas Fúngicas/biossíntese , Genoma Fúngico , Proteínas de Choque Térmico/biossíntese , Temperatura Alta , Mitocôndrias/química , Mitocôndrias/genética , Biossíntese de Proteínas , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/fisiologiaRESUMO
Subunit 9 of mitochondrial ATPase (Su9) is synthesized in reticulocyte lysates programmed with Neurospora poly A-RNA, and in a Neurospora cell free system as a precursor with a higher apparent molecular weight than the mature protein (Mr 16,400 vs. 10,500). The RNA which directs the synthesis of Su9 precursor is associated with free polysomes. The precursor occurs as a high molecular weight aggregate in the postribosomal supernatant of reticulocyte lysates. Transfer in vitro of the precursor into isolated mitochondria is demonstrated. This process includes the correct proteolytic cleavage of the precursor to the mature form. After transfer, the protein acquires the following properties of the assembled subunit: it is resistant to added protease, it is soluble in chloroform/methanol, and it can be immunoprecipitated with antibodies to F1-ATPase. The precursor to Su9 is also detected in intact cells after pulse labeling. Processing in vivo takes place posttranslationally. It is inhibited by the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). A hypothetical mechanism is discussed for the intracellular transfer of Su9. It entails synthesis on free polysomes, release of the precursor into the cytosol, recognition by a receptor on the mitochondrial surface, and transfer into the inner mitochondrial membrane, which is accompanied by proteolytic cleavage and which depends on an electrical potential across the inner mitochondrial membrane.
Assuntos
Adenosina Trifosfatases/biossíntese , Precursores Enzimáticos/biossíntese , Mitocôndrias/enzimologia , Neurospora crassa/enzimologia , Neurospora/enzimologia , Adenosina Trifosfatases/análise , Adenosina Trifosfatases/imunologia , Anticorpos , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Sistema Livre de Células , Polirribossomos/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Mitochondrial membrane fusion is a process essential for the maintenance of the structural integrity of the organelle. Since mitochondria are bounded by a double membrane, they face the challenge of fusing four membranes in a coordinated manner. We provide evidence that this is achieved by coupling of the mitochondrial outer and inner membranes by the mitochondrial fusion machinery. Fzo1, the first known mediator of mitochondrial fusion, spans the outer membrane twice, exposing a short loop to the intermembrane space. The presence of the intermembrane space segment is required for the localization of Fzo1 in sites of tight contact between the mitochondrial outer and inner membranes. Mutations in the intermembrane space domain of yeast Fzo1 relieve the association with the inner membrane. This results in a loss of function of the protein in vivo. We propose that the mitochondrial fusion machinery forms membrane contact sites that mediate mitochondrial fusion. A fusion machinery that is in contact with both mitochondrial membranes appears to be functionally important for coordinated fusion of four mitochondrial membranes.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Membranas Intracelulares/fisiologia , Fusão de Membrana , Proteínas de Membrana/metabolismo , Mitocôndrias/fisiologia , Polaridade Celular , Citosol , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , GTP Fosfo-Hidrolases/genética , Proteínas de Membrana/genética , Proteínas Mitocondriais , Modelos Biológicos , Mutação , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae , Partículas Submitocôndricas , LevedurasRESUMO
Mitochondrial heat shock protein 70 (mt-Hsp70) has been shown to play an important role in facilitating import into, as well as folding and assembly of nuclear-encoded proteins in the mitochondrial matrix. Here, we describe a role for mt-Hsp70 in chaperoning proteins encoded by mitochondrial DNA and synthesized within mitochondria. The availability of mt-Hsp70 function influences the pattern of proteins synthesized in mitochondria of yeast both in vivo and in vitro. In particular, we show that mt-Hsp70 acts in maintaining the var1 protein, the only mitochondrially encoded subunit of mitochondrial ribosomes, in an assembly competent state, especially under heat stress conditions. Furthermore, mt-Hsp70 helps to facilitate assembly of mitochondrially encoded subunits of the ATP synthase complex. By interacting with the ATP-ase 9 oligomer, mt-Hsp70 promotes assembly of ATP-ase 6, and thereby protects the latter protein from proteolytic degradation. Thus mt-Hsp70 by acting as a chaperone for proteins encoded by the mitochondrial DNA, has a critical role in the assembly of supra-molecular complexes.
Assuntos
DNA Mitocondrial/metabolismo , Proteínas Fúngicas/biossíntese , Proteínas de Choque Térmico HSP70/biossíntese , Mitocôndrias/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Fúngicas/isolamento & purificação , Proteínas de Choque Térmico HSP70/isolamento & purificação , Proteínas de Choque Térmico HSP70/metabolismo , Substâncias Macromoleculares , Modelos Biológicos , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , ATPases Translocadoras de Prótons/biossíntese , ATPases Translocadoras de Prótons/isolamento & purificação , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , TemperaturaRESUMO
The novel genetic method of "sheltered RIP" (repeat induced point mutation) was used to generate a Neurospora crassa mutant in which MOM19, a component of the protein import machinery of the mitochondrial outer membrane, can be depleted. Deficiency in MOM19 resulted in a severe growth defect, but the cells remained viable. The number of mitochondrial profiles was not grossly changed, but mutant mitochondria were highly deficient in cristae membranes, cytochromes, and protein synthesis activity. Protein import into isolated mutant mitochondria was decreased by factors of 6 to 30 for most proteins from all suborganellar compartments. Proteins like the ADP/ATP carrier, MOM19, and cytochrome c, whose import into wild-type mitochondria occurs independently of MOM19 became imported normally showing that the reduced import activities are solely caused by a lack of MOM19. Depletion of MOM19 reveals a close functional relationship between MOM19 and MOM22, since loss of MOM19 led to decreased levels of MOM22 and reduced protein import through MOM22. Furthermore, MOM72 does not function as a general backup receptor for MOM19 suggesting that these two proteins have distinct precursor specificities. These findings demonstrate that the import receptor MOM19 fulfills an important role in the biogenesis of mitochondria and that it is essential for the formation of mitochondria competent in respiration and phosphorylation.
Assuntos
Proteínas de Membrana Transportadoras , Mitocôndrias/metabolismo , Neurospora crassa/metabolismo , Receptores de Superfície Celular , Receptores Citoplasmáticos e Nucleares/metabolismo , Citocromos/biossíntese , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Cinética , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Mitocôndrias/ultraestrutura , Neurospora crassa/genética , Neurospora crassa/crescimento & desenvolvimento , Mutação Puntual , Receptores Citoplasmáticos e Nucleares/biossíntese , Receptores Citoplasmáticos e Nucleares/genética , Sequências Repetitivas de Ácido NucleicoRESUMO
The precursor of porin, a mitochondrial outer membrane protein, competes for the import of precursors destined for the three other mitochondrial compartments, including the Fe/S protein of the bc1-complex (intermembrane space), the ADP/ATP carrier (inner membrane), subunit 9 of the F0-ATPase (inner membrane), and subunit beta of the F1-ATPase (matrix). Competition occurs at the level of a common site at which precursors are inserted into the outer membrane. Protease-sensitive binding sites, which act before the common insertion site, appear to be responsible for the specificity and selectivity of mitochondrial protein uptake. We suggest that distinct receptor proteins on the mitochondrial surface specifically recognize precursor proteins and transfer them to a general insertion protein component (GIP) in the outer membrane. Beyond GIP, the import pathways diverge, either to the outer membrane or to translocation contact-sites, and then subsequently to the other mitochondrial compartments.
Assuntos
Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Neurospora crassa/metabolismo , Neurospora/metabolismo , Porinas , Precursores de Proteínas/metabolismo , Sítios de Ligação , Ligação Competitiva , Membrana Celular/metabolismo , Neurospora crassa/ultraestrutura , Canais de Ânion Dependentes de VoltagemRESUMO
Passage of precursor proteins through translocation contact sites of mitochondria was investigated by studying the import of a fusion protein consisting of the NH2-terminal 167 amino acids of yeast cytochrome b2 precursor and the complete mouse dihydrofolate reductase. Isolated mitochondria of Neurospora crassa readily imported the fusion protein. In the presence of methotrexate import was halted and a stable intermediate spanning both mitochondrial membranes at translocation contact sites accumulated. The complete dihydrofolate reductase moiety in this intermediate was external to the outer membrane, and the 136 amino acid residues of the cytochrome b2 moiety remaining after cleavage by the matrix processing peptidase spanned both outer and inner membranes. Removal of methotrexate led to import of the intermediate retained at the contact site into the matrix. Thus unfolding at the surface of the outer mitochondrial membrane is a prerequisite for passage through translocation contact sites. The membrane-spanning intermediate was used to estimate the number of translocation sites. Saturation was reached at 70 pmol intermediate per milligram of mitochondrial protein. This amount of translocation intermediates was calculated to occupy approximately 1% of the total surface of the outer membrane. The morphometrically determined area of close contact between outer and inner membranes corresponded to approximately 7% of the total outer membrane surface. Accumulation of the intermediate inhibited the import of other precursor proteins suggesting that different precursor proteins are using common translocation contact sites. We conclude that the machinery for protein translocation into mitochondria is present at contact sites in limited number.
Assuntos
Precursores Enzimáticos/metabolismo , L-Lactato Desidrogenase/metabolismo , Mitocôndrias/metabolismo , Neurospora crassa/metabolismo , Neurospora/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Precursores Enzimáticos/genética , Cinética , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase (Citocromo) , Camundongos , Microscopia Eletrônica , Mitocôndrias/ultraestrutura , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/enzimologia , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismoRESUMO
Tom40 is the main component of the preprotein translocase of the outer membrane of mitochondria (TOM complex). We have isolated Tom40 of Neurospora crassa by removing the receptor Tom22 and the small Tom components Tom6 and Tom7 from the purified TOM core complex. Tom40 is organized in a high molecular mass complex of approximately 350 kD. It forms a high conductance channel. Mitochondrial presequence peptides interact specifically with Tom40 reconstituted into planar lipid membranes and decrease the ion flow through the pores in a voltage-dependent manner. The secondary structure of Tom40 comprises approximately 31% beta-sheet, 22% alpha-helix, and 47% remaining structure as determined by circular dichroism measurements and Fourier transform infrared spectroscopy. Electron microscopy of purified Tom40 revealed particles primarily with one center of stain accumulation. They presumably represent an open pore with a diameter of approximately 2.5 nm, similar to the pores found in the TOM complex. Thus, Tom40 is the core element of the TOM translocase; it forms the protein-conducting channel in an oligomeric assembly.
Assuntos
Proteínas Fúngicas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Mitocôndrias/metabolismo , Proteínas de Saccharomyces cerevisiae , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/ultraestrutura , Membranas Intracelulares/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/ultraestrutura , Proteínas de Transporte da Membrana Mitocondrial , Neurospora crassa/metabolismo , Estrutura Secundária de ProteínaRESUMO
The biogenesis of mitochondria requires the integration of many proteins into the inner membrane from the matrix side. The inner membrane protein Oxa1 plays an important role in this process. We identified Mba1 as a second mitochondrial component that is required for efficient protein insertion. Like Oxa1, Mba1 specifically interacts both with mitochondrial translation products and with conservatively sorted, nuclear-encoded proteins during their integration into the inner membrane. Oxa1 and Mba1 overlap in function and substrate specificity, but both can act independently of each other. We conclude that Mba1 is part of the mitochondrial protein export machinery and represents the first component of a novel Oxa1-independent insertion pathway into the mitochondrial inner membrane.
Assuntos
Proteínas Fúngicas/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Divisão Celular , Núcleo Celular/genética , Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas Fúngicas/genética , Teste de Complementação Genética , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Proteínas de Membrana/genética , Mitocôndrias/química , Mitocôndrias/enzimologia , Proteínas Mitocondriais , Mutação/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Testes de Precipitina , Ligação Proteica , Biossíntese de Proteínas , Transporte Proteico , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Especificidade por SubstratoRESUMO
With vital yeast cells, a hybrid protein consisting of the amino-terminal third of the precursor to cytochrome b2 and of the entire dihydrofolate reductase was arrested on the import pathway into mitochondria. Accumulation of the protein in the mitochondrial membranes was achieved by inducing a stable tertiary structure of the dihydrofolate reductase domain. Thereby, three salient features of mitochondrial protein uptake in vivo were demonstrated: its posttranslational character; the requirement for unfolding of precursors; and import through translocation contact sites. The permanent occupation of translocation sites by the fusion protein inhibited the import of other precursors; it did, however, not lead to leakage of mitochondrial ions, implying the existence of a channel that is sealed around the membrane spanning polypeptide segment.
Assuntos
Mitocôndrias/metabolismo , Precursores de Proteínas/metabolismo , Aminopterina/farmacologia , Transporte Biológico , Membranas Intracelulares/metabolismo , Cinética , L-Lactato Desidrogenase/metabolismo , L-Lactato Desidrogenase (Citocromo) , Potenciais da Membrana , Conformação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Tetra-Hidrofolato Desidrogenase/metabolismoRESUMO
Translocation of nuclear-encoded preproteins across the outer membrane of mitochondria is mediated by the multicomponent transmembrane TOM complex. We have isolated the TOM core complex of Neurospora crassa by removing the receptors Tom70 and Tom20 from the isolated TOM holo complex by treatment with the detergent dodecyl maltoside. It consists of Tom40, Tom22, and the small Tom components, Tom6 and Tom7. This core complex was also purified directly from mitochondria after solubilization with dodecyl maltoside. The TOM core complex has the characteristics of the general insertion pore; it contains high-conductance channels and binds preprotein in a targeting sequence-dependent manner. It forms a double ring structure that, in contrast to the holo complex, lacks the third density seen in the latter particles. Three-dimensional reconstruction by electron tomography exhibits two open pores traversing the complex with a diameter of approximately 2.1 nm and a height of approximately 7 nm. Tom40 is the key structural element of the TOM core complex.
Assuntos
Proteínas de Transporte/química , Membranas Intracelulares/enzimologia , Proteínas de Membrana/química , Mitocôndrias/enzimologia , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Proteínas de Transporte/ultraestrutura , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/ultraestrutura , Membranas Intracelulares/metabolismo , Membranas Intracelulares/fisiologia , Membranas Intracelulares/ultraestrutura , Canais Iônicos/química , Canais Iônicos/metabolismo , Canais Iônicos/fisiologia , Canais Iônicos/ultraestrutura , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Mitocôndrias/ultraestrutura , Neurospora crassa/enzimologia , Neurospora crassa/metabolismo , Neurospora crassa/fisiologia , Neurospora crassa/ultraestrutura , Ligação Proteica , Precursores de Proteínas/metabolismo , Precursores de Proteínas/ultraestruturaRESUMO
We have identified the yeast homologue of Neurospora crassa MOM72, the mitochondrial import receptor for the ADP/ATP carrier (AAC), by functional studies and by cDNA sequencing. Mitochondria of a yeast mutant in which the gene for MOM72 was disrupted were impaired in specific binding and import of AAC. Unexpectedly, we found a residual, yet significant import of AAC into mitochondria lacking MOM72 that occurred via the receptor MOM19. We conclude that both MOM72 and MOM19 can direct AAC into mitochondria, albeit with different efficiency. Moreover, the precursor of MOM72 apparently does not require a positively charged sequence at the extreme amino terminus for targeting to mitochondria.
Assuntos
Proteínas Fúngicas , Proteínas de Membrana , Mitocôndrias/enzimologia , Translocases Mitocondriais de ADP e ATP/genética , Neurospora crassa/genética , Processamento de Proteína Pós-Traducional , Receptores de Superfície Celular/genética , Receptores Citoplasmáticos e Nucleares , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Fúngico/genética , Genes Fúngicos , Cinética , Translocases Mitocondriais de ADP e ATP/metabolismo , Proteínas de Transporte da Membrana Mitocondrial , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Dados de Sequência Molecular , Peso Molecular , Neurospora crassa/enzimologia , Receptores de Superfície Celular/isolamento & purificação , Receptores de Superfície Celular/metabolismo , Saccharomyces cerevisiae/enzimologia , Homologia de Sequência do Ácido NucleicoRESUMO
Porin, also termed the voltage-dependent anion channel, is the most abundant protein of the mitochondrial outer membrane. The process of import and assembly of the protein is known to be dependent on the surface receptor Tom20, but the requirement for other mitochondrial proteins remains controversial. We have used mitochondria from Neurospora crassa and Saccharomyces cerevisiae to analyze the import pathway of porin. Import of porin into isolated mitochondria in which the outer membrane has been opened is inhibited despite similar levels of Tom20 as in intact mitochondria. A matrix-destined precursor and the porin precursor compete for the same translocation sites in both normal mitochondria and mitochondria whose surface receptors have been removed, suggesting that both precursors utilize the general import pore. Using an assay established to monitor the assembly of in vitro-imported porin into preexisting porin complexes we have shown that besides Tom20, the biogenesis of porin depends on the central receptor Tom22, as well as Tom5 and Tom7 of the general import pore complex (translocase of the outer mitochondrial membrane [TOM] core complex). The characterization of two new mutant alleles of the essential pore protein Tom40 demonstrates that the import of porin also requires a functional Tom40. Moreover, the porin precursor can be cross-linked to Tom20, Tom22, and Tom40 on its import pathway. We conclude that import of porin does not proceed through the action of Tom20 alone, but requires an intact outer membrane and involves at least four more subunits of the TOM machinery, including the general import pore.