RESUMO
Two members of the heat shock protein 70 kDa (Hsp70) family, Ssc1 and Ssq1, perform important functions in the mitochondrial matrix. The essential Ssc1 is an abundant ATP-binding protein required for both import and folding of mitochondrial proteins. The function of Ssc1 is supported by an interaction with the preprotein translocase subunit Tim44, the cochaperone Mdj1, and the nucleotide exchange factor Mge1. In contrast, only limited information is available on Ssq1. So far, a basic characterization of Ssq1 has demonstrated its involvement in the maintenance of mitochondrial DNA, the maturation of the yeast frataxin (Yfh1) after import, and assembly of the mitochondrial Fe/S cluster. Here, we analyzed the biochemical properties and the interaction partners of Ssq1 in detail. Ssq1 showed typical chaperone properties by binding to unfolded substrate proteins in an ATP-regulated manner. Ssq1 was able to form a specific complex with the nucleotide exchange factor Mge1. In particular, complex formation in organello was enhanced significantly when Ssc1 was inactivated selectively. However, even under these conditions, no interaction of Ssq1 with the two other mitochondrial Hsp70-cochaperones, Tim44 and Mdj1, was observed. The Ssq1-Mge1 interaction showed a lower overall stability but the same characteristic nucleotide-dependence as the Ssc1-Mge1 interaction. A quantitative analysis of the interaction properties indicated a competition of Ssq1 with Ssc1 for binding to Mge1. Perturbation of Mge1 function or amounts resulted in direct effects on Ssq1 activity in intact mitochondria. We conclude that mitochondria represent the unique case where two Hsp70s compete for the interaction with one nucleotide exchange factor.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Membrana Transportadoras , Proteínas de Transporte da Membrana Mitocondrial , Proteínas Mitocondriais/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Ligação Competitiva , ATPases Transportadoras de Cálcio/química , Proteínas de Transporte/química , Sequência Conservada , Proteínas Fúngicas/química , Proteínas de Choque Térmico HSP40 , Proteínas de Choque Térmico HSP70 , Proteínas de Choque Térmico/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/química , Mitocôndrias/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Modelos Moleculares , Chaperonas Moleculares/química , Dados de Sequência Molecular , Testes de Precipitina , Ligação Proteica , Conformação Proteica , Precursores de Proteínas/metabolismo , Transporte Proteico , Saccharomyces cerevisiae/citologiaRESUMO
The mitochondrial heat shock protein Hsp70 (mtHsp70) is essential for driving translocation of preproteins into the matrix. Two models, trapping and pulling by mtHsp70, are discussed, but positive evidence for either model has not been found so far. We have analyzed a mutant mtHsp70, Ssc1-2, that shows a reduced interaction with the membrane anchor Tim44, but an enhanced trapping of preproteins. Unexpectedly, at a low inner membrane potential, ssc1-2 mitochondria imported loosely folded preproteins more efficiently than wild-type mitochondria. The import of a tightly folded preprotein, however, was not increased in ssc1-2 mitochondria. Thus, enhanced trapping by mtHsp70 stimulates the import of loosely folded preproteins and reduces the dependence on the import-driving activity of the membrane potential, directly demonstrating that trapping is one of the molecular mechanisms of mtHsp70 action.
Assuntos
Membrana Celular/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico , Proteínas de Membrana Transportadoras , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial , Proteínas de Saccharomyces cerevisiae , ATPases Transportadoras de Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Cisplatino/farmacologia , Reagentes de Ligações Cruzadas/farmacologia , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Proteínas Fúngicas/metabolismo , Potenciais da Membrana , Proteínas de Membrana/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Chaperonas Moleculares/metabolismo , Mutação , Testes de Precipitina , Transporte Proteico , Saccharomyces cerevisiae/metabolismo , Fatores de TempoRESUMO
Most mitochondrial proteins are nuclear-encoded and synthesised as preproteins on polysomes in the cytosol. They must be targeted to and translocated into mitochondria. Newly synthesised preproteins interact with cytosolic factors until their recognition by receptors on the surface of mitochondria. Import into or across the outer membrane is mediated by a dynamic protein complex coined the translocase of the outer membrane (TOM). Preproteins that are imported into the matrix or inner membrane of mitochondria require the action of one of two translocation complexes of the inner membrane (TIMs). The import pathway of preproteins is predetermined by their intrinsic targeting and sorting signals. Energy input in the form of ATP and the electrical gradient across the inner membrane is required for protein translocation into mitochondria. Newly imported proteins may require molecular chaperones for their correct folding.
Assuntos
Proteínas de Bactérias , Proteínas de Escherichia coli , Proteínas de Membrana Transportadoras , Mitocôndrias/metabolismo , Transporte Proteico , Adenosina Trifosfatases/metabolismo , Animais , Proteínas de Transporte/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Precursores de Proteínas/metabolismo , Canais de Translocação SEC , Proteínas SecAAssuntos
Mitocôndrias/metabolismo , Sinais Direcionadores de Proteínas/fisiologia , Transporte Proteico/fisiologia , Proteínas/metabolismo , Immunoblotting , Potenciais da Membrana/fisiologia , Mitocôndrias/química , Reação em Cadeia da Polimerase , Biossíntese de Proteínas , Precursores de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transcrição GênicaRESUMO
Unfolding is an essential process during translocation of preproteins into mitochondria; however, controversy exists as to whether mitochondria play an active role in unfolding. We have established an in vitro system with a kinetic saturation of the mitochondrial import machinery, yielding translocation rates comparable to in vivo import rates. Preproteins with short N-terminal segments in front of a folded domain show a characteristic delay of the onset of translocation (lag phase) although the maximal import rate is similar to that of longer preproteins. The lag phase is shortened by extending the N-terminal segment to improve the accessibility to matrix heat shock protein 70 and abolished by unfolding of the preprotein. A mutant mtHsp70 defective in binding to the inner membrane prolongs the lag phase and reduces the translocation activity. A direct comparison of the rate of spontaneous unfolding in solution with that during translocation demonstrates that unfolding by mitochondria is significantly faster, proving an active unfolding process. We conclude that access of mtHsp70 to N-terminal preprotein segments is critical for active unfolding and initiation of translocation.
Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Mitocôndrias/metabolismo , Dobramento de Proteína , Precursores de Proteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Animais , Eletroforese em Gel de Poliacrilamida , Cinética , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , L-Lactato Desidrogenase (Citocromo) , Camundongos , Mitocôndrias/genética , Mutação , Desnaturação Proteica/efeitos dos fármacos , Precursores de Proteínas/química , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo , Ureia/farmacologiaRESUMO
The biogenesis of mitochondria requires the translocation of most mitochondrial proteins across two biological membranes. Mitochondrial preproteins are synthesized in the cytosol carrying targeting information, that is recognized by specific receptor proteins. The precursor polypeptides are transported across both mitochondrial membranes via three large integral membrane protein complexes forming specialized preprotein translocases. A soluble protein complex in the matrix provides the ATP-dependent translocation force, responsible for the movement and unfolding of the bulk polypeptide chain. After the removal of the targeting sequence, imported proteins fold into their native conformation with the help of chaperone proteins in the mitochondrial matrix.
Assuntos
Mitocôndrias/fisiologia , Transporte Proteico , Transporte Biológico Ativo , Proteínas de Transporte/metabolismo , Proteínas de Choque Térmico/metabolismo , Membranas Intracelulares/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismoAssuntos
Citrato (si)-Sintase/química , Citrato (si)-Sintase/metabolismo , Eletroforese em Gel de Poliacrilamida/métodos , Mitocôndrias/enzimologia , Saccharomyces cerevisiae/enzimologia , Citrato (si)-Sintase/biossíntese , Dimerização , Eletroforese em Gel Bidimensional , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Cinética , Mitocôndrias/metabolismo , Transporte Proteico , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Radioisótopos de EnxofreRESUMO
Mitochondrial proteins are synthesized as precursor proteins in the cytosol and are posttranslationally imported into the organelle. A complex system of translocation machineries recognizes and transports the precursor polypeptide across the mitochondrial membranes. Energy for the translocation process is mainly supplied by the mitochondrial membrane potential (deltapsi) and the hydrolysis of ATP. Mitochondrial Hsp70 (mtHsp70) has been identified as the major ATPase driving the membrane transport of the precursor polypeptides into the mitochondrial matrix. Together with the partner proteins Tim44 and Mge1, mtHsp70 forms an import motor complex interacting with the incoming preproteins at the inner face of the inner membrane. This import motor complex drives the movement of the polypeptides in the translocation channel and the unfolding of carboxy-terminal parts of the preproteins on the outside of the outer membrane. Two models of the molecular mechanism of mtHsp70 during polypeptide translocation are discussed. In the 'trapping' model, precursor movement is generated by Brownian movement of the polypeptide chain in the translocation pore. This random movement is made vectorial by the interaction with mtHsp70 in the matrix. The detailed characterization of conditional mutants of the import motor complex provides the basis for an extended model. In this 'pulling' model, the attachment of mtHsp70 at the inner membrane via Tim44 and a conformational change induced by ATP results in the generation of an inward-directed force on the bound precursor polypeptide. This active role of the import motor complex is necessary for the translocation of proteins containing tightly folded domains. We suggest that both mechanisms complement each other to reach a high efficiency of preprotein import.
Assuntos
Mitocôndrias/metabolismo , Proteínas Motores Moleculares/metabolismo , Proteínas/fisiologia , Animais , Proteínas de Choque Térmico/metabolismo , HumanosRESUMO
The import motor for preproteins that are targeted into the mitochondrial matrix consists of the matrix heat shock protein Hsp70 (mtHsp70) and the translocase subunit Tim44 of the inner membrane. mtHsp70 interacts with Tim44 in an ATP-dependent reaction cycle, binds to preproteins in transit, and drives their translocation into the matrix. While different functional mechanisms are discussed for the mtHsp70-Tim44 machinery, little is known about the actual mode of interaction of both proteins. Here, we have addressed which of the three Hsp70 regions, the ATPase domain, the peptide binding domain, or the carboxy-terminal segment, are required for the interaction with Tim44. By two independent means, a two-hybrid system and coprecipitation of mtHsp70 constructs imported into mitochondria, we show that the ATPase domain interacts with Tim44, although with a reduced efficiency compared to the full-length mtHsp70. The interaction of the ATPase domain with Tim44 is ATP sensitive. The peptide binding domain and carboxy-terminal segment are unable to bind to Tim44 in the absence of the ATPase domain, but both regions enhance the interaction with Tim44 in the presence of the ATPase domain. We conclude that the ATPase domain of mtHsp70 is essential for and directly interacts with Tim44, clearly separating the mtHsp70-Tim44 interaction from the mtHsp70-substrate interaction.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial , Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfatases/metabolismo , Transporte Biológico , Proteínas de Transporte/genética , Proteínas de Choque Térmico HSP70/genética , Proteínas de Membrana/genética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Ligação Proteica , Saccharomyces cerevisiaeRESUMO
Protein unfolding is a key step in the import of some proteins into mitochondria and chloroplasts and in the degradation of regulatory proteins by ATP-dependent proteases. In contrast to protein folding, the reverse process has remained largely uninvestigated until now. This review discusses recent discoveries on the mechanism of protein unfolding during translocation into mitochondria. The mitochondria can actively unfold preproteins by unraveling them from the N-terminus. The central component of the mitochondrial import motor, the matrix heat shock protein 70, functions by both pulling and holding the preproteins.
Assuntos
Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/metabolismo , Mitocôndrias/fisiologia , Dobramento de Proteína , Trifosfato de Adenosina/metabolismo , Animais , Proteínas de Bactérias , Citosol/metabolismo , Modelos Biológicos , Ligação Proteica , Estrutura Terciária de Proteína , Ribonucleases/química , Ribonucleases/metabolismoRESUMO
Mitochondrial biogenesis utilizes a complex proteinaceous machinery for the import of cytosolically synthesized preproteins. At least three large multisubunit protein complexes, one in the outer membrane and two in the inner membrane, have been identified. These translocase complexes cooperate with soluble proteins from the cytosol, the intermembrane space and the matrix. The translocation of presequence-containing preproteins through the outer membrane channel includes successive electrostatic interactions of the charged mitochondrial targeting sequence with a chain of import components. Translocation across the inner mitochondrial membrane utilizes the energy of the proton motive force of the inner membrane and the hydrolysis of ATP. The matrix chaperone system of the mitochondrial heat shock protein 70 forms an ATP-dependent import motor by interaction with the polypeptide chain in transit and components of the inner membrane translocase. The precursors of integral inner membrane proteins of the metabolite carrier family interact with newly identified import components of the intermembrane space and are inserted into the inner membrane by a second translocase complex. A comparison of the full set of import components between the yeast Sacccharomyces cerevisiae and the nematode Caenorhabditis elegans demonstrates an evolutionary conservation of most components of the mitochondrial import machinery with a possible greater divergence for the import pathway of the inner membrane carrier proteins.
Assuntos
Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas de Saccharomyces cerevisiae , Animais , Transporte Biológico , Caenorhabditis elegans , Proteínas de Transporte/metabolismo , Citosol/metabolismo , Evolução Molecular , Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Potenciais da Membrana , Proteínas de Transporte da Membrana Mitocondrial , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Saccharomyces cerevisiaeRESUMO
Tim44 is a protein of the mitochondrial inner membrane and serves as an adaptor protein for mtHsp70 that drives the import of preproteins in an ATP-dependent manner. In this study we have modified the interaction of Tim44 with mtHsp70 and characterized the consequences for protein translocation. By deletion of an 18-residue segment of Tim44 with limited similarity to J-proteins, the binding of Tim44 to mtHsp70 was weakened. We found that in the yeast Saccharomyces cerevisiae the deletion of this segment is lethal. To investigate the role of the 18-residue segment, we expressed Tim44Delta18 in addition to the endogenous wild-type Tim44. Tim44Delta18 is correctly targeted to mitochondria and assembles in the inner membrane import site. The coexpression of Tim44Delta18 together with wild-type Tim44, however, does not stimulate protein import, but reduces its efficiency. In particular, the promotion of unfolding of preproteins during translocation is inhibited. mtHsp70 is still able to bind to Tim44Delta18 in an ATP-regulated manner, but the efficiency of interaction is reduced. These results suggest that the J-related segment of Tim44 is needed for productive interaction with mtHsp70. The efficient cooperation of mtHsp70 with Tim44 facilitates the translocation of loosely folded preproteins and plays a crucial role in the import of preproteins which contain a tightly folded domain.
Assuntos
Proteínas de Transporte/fisiologia , Sobrevivência Celular/fisiologia , Proteínas de Choque Térmico HSP70/fisiologia , Proteínas de Membrana/fisiologia , Mitocôndrias/fisiologia , Proteínas de Transporte da Membrana Mitocondrial , Mutação , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiologia , Sequência de Aminoácidos , Transporte Biológico , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Dados de Sequência Molecular , Saccharomyces cerevisiae/ultraestruturaRESUMO
Mitochondrial heat shock protein 70 (mtHsp70) functions in unfolding, translocation, and folding of imported proteins. Controversial models of mtHsp70 action have been discussed: (1) physical trapping of preproteins is sufficient to explain the various mtHsp70 functions, and (2) unfolding of preproteins requires an active motor function of mtHsp70 ("pulling"). Intragenic suppressors of a mutant mtHsp70 separate two functions: a nonlethal folding defect caused by enhanced trapping of preproteins, and a conditionally lethal unfolding defect caused by an impaired interaction of mtHsp70 with the membrane anchor Tim44. Even enhanced trapping in wild-type mitochondria does not generate a pulling force. The motor function of mtHsp70 cannot be explained by passive trapping alone but includes an essential ATP-dependent interaction with Tim44 to generate a pulling force and unfold preproteins.
Assuntos
ATPases Transportadoras de Cálcio , Proteínas de Choque Térmico HSP70/metabolismo , Mitocôndrias/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Precursores de Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Cinética , Modelos Moleculares , Chaperonas Moleculares/genética , Dobramento de Proteína , Precursores de Proteínas/química , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/genética , Supressão Genética , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/metabolismo , Valinomicina/farmacologiaRESUMO
The ADP/ATP carrier (AAC) of the mitochondrial inner membrane is synthesized in the cytosol without a cleavable presequence. The preprotein preferentially binds to the mitochondrial surface receptor Tom70 and joins the import pathway of presequence-carrying preproteins at the cis side of the outer membrane. Little is known about the translocation of the AAC across the outer membrane and where its import route separates from that of cleavable preproteins. Here we have characterized a translocation intermediate of AAC during transfer across the outer membrane. The major portion of the preprotein is exposed to the intermembrane space, while a short segment is still accessible to externally added protease. This intermediate can be quantitatively chased to the fully imported form in the inner membrane. Its accumulation depends on Tom7, but not on the intermembrane space domain of Tom22 in contrast to cleavable preproteins. Moreover, opening of the intermembrane space inhibits the import of AAC, but not that of cleavable preproteins into mitoplasts. We conclude that the import route of AAC diverges from the general import pathway of cleavable preproteins already at the trans side of the outer membrane.
Assuntos
Proteínas de Membrana Transportadoras , Mitocôndrias/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Receptores de Superfície Celular , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Proteínas de Transporte/metabolismo , Proteínas Fúngicas/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Transporte da Membrana Mitocondrial , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
The dynamic vesicle transport processes at the late-Golgi compartment of Saccharomyces cerevisiae (TGN) require dedicated mechanisms for correct localization of resident membrane proteins. In this study, we report the identification of a new gene, GRD19, involved in the localization of the model late-Golgi membrane protein A-ALP (consisting of the cytosolic domain of dipeptidyl aminopeptidase A [DPAP A] fused to the transmembrane and lumenal domains of the alkaline phosphatase [ALP]), which localizes to the yeast TGN. A grd19 null mutation causes rapid mislocalization of the late-Golgi membrane proteins A-ALP and Kex2p to the vacuole. In contrast to previously identified genes involved in late-Golgi membrane protein localization, grd19 mutations cause only minor effects on vacuolar protein sorting. The recycling of the carboxypeptidase Y sorting receptor, Vps10p, between the TGN and the prevacuolar compartment is largely unaffected in grd19Delta cells. Kinetic assays of A-ALP trafficking indicate that GRD19 is involved in the process of retrieval of A-ALP from the prevacuolar compartment. GRD19 encodes a small hydrophilic protein with a predominantly cytosolic distribution. In a yeast mutant that accumulates an exaggerated form of the prevacuolar compartment (vps27), Grd19p was observed to localize to this compartment. Using an in vitro binding assay, Grd19p was found to interact physically with the cytosolic domain of DPAP A. We conclude that Grd19p is a component of the retrieval machinery that functions by direct interaction with the cytosolic tails of certain TGN membrane proteins during the sorting/budding process at the prevacuolar compartment.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Transporte/fisiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/fisiologia , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Pró-Proteína Convertases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Proteínas de Transporte Vesicular , Fosfatase Alcalina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Carboxipeptidases/metabolismo , Proteínas de Transporte/análise , Proteínas de Transporte/química , Catepsina A , Clonagem Molecular , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Proteínas Fúngicas/análise , Proteínas Fúngicas/química , Genes Fúngicos , Membranas Intracelulares/metabolismo , Dados de Sequência Molecular , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Subtilisinas/metabolismoRESUMO
The mitochondrial heat shock protein Hsp70 is essential for import of nuclear-encoded proteins, involved in both unfolding and membrane translocation of preproteins. mtHsp70 interacts reversibly with Tim44 of the mitochondrial inner membrane, yet the role of this interaction is unknown. We analysed this role by using two yeast mutants of mtHsp70 that differentially influenced its interaction with Tim44. One mutant mtHsp70 (Ssc1-2p) efficiently bound preproteins, but did not show a detectable complex formation with Tim44; the mitochondria imported loosely folded preproteins with wild-type kinetics, yet were impaired in unfolding of preproteins. The other mutant Hsp70 (Ssc1-3p') bound both Tim44 and preproteins, but the mitochondria did not import folded polypeptides and were impaired in import of unfolded preproteins; Ssc1-3p' was defective in its ATPase domain and did not undergo a nucleotide-dependent conformational change, resulting in permanent binding to Tim44. The following conclusions are suggested. (i) The import of loosely folded polypeptides (translocase function of mtHsp70) does not depend on formation of a detectable Hsp70-Tim44 complex. Two explanations are possible: a trapping mechanism by soluble mtHsp70, or a weak/very transient interaction of Ssc1-2p with Tim44 that leads to a weak force generation sufficient for import of loosely folded, but not folded, polypeptides. (ii) Import of folded preproteins (unfoldase function of mtHsp70) involves a reversible nucleotide-dependent interaction of mtHsp70 with Tim44, including a conformational change in mtHsp70. This is consistent with a model that the dynamic interaction of mtHsp70 with Tim44 generates a pulling force on preproteins which supports unfolding during translocation.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Transporte da Membrana Mitocondrial , Precursores de Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae , Nucleotídeos de Adenina/metabolismo , Trifosfato de Adenosina/metabolismo , Transporte Biológico Ativo , Proteínas Fúngicas/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Ligação Proteica , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiaeRESUMO
The mitochondrial heat shock protein Hsp78 is a member of the Hsp104/Clp family with unknown function. Saccharomyces cerevisiae deletion mutants of HSP78 show wild-type like growth. We report that deletion of the HSP78 gene in yeast strains with point mutations in the SSC1 gene (encoding matrix Hsp70) led to loss of mitochondrial DNA, indicating that at least one of the heat shock proteins Hsp78 and mt-Hsp70 is needed to maintain a rho+ state of the mitochondrial genome. Mitochondria isolated from these double mutants had a strongly reduced membrane potential, explaining defects in the rate of preprotein import. The lack of Hsp78 led to aggregation of the mutant mt-Hsp70 while other matrix chaperones stayed soluble. We conclude that Hsp78 is required to keep mutant forms of mt-Hsp70 soluble and suggest a cooperation of Hsp78 and mt-Hsp70 in maintenance of essential mitochondrial functions.
Assuntos
Proteínas de Escherichia coli , Matriz Extracelular/química , Proteínas Fúngicas/genética , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico/genética , Mitocôndrias/fisiologia , Proteínas de Saccharomyces cerevisiae , Divisão Celular , Membrana Celular , Endopeptidase Clp , Proteínas Fúngicas/metabolismo , Deleção de Genes , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Potenciais da Membrana , Mitocôndrias/química , Mitocôndrias/ultraestrutura , Mutação Puntual , Saccharomyces cerevisiae/química , Homologia de Sequência de AminoácidosRESUMO
Interaction of preproteins with the heat shock protein Hsp70 in the mitochondrial matrix is required for driving protein transport across the mitochondrial inner membrane. Binding of mt-Hsp70 to the protein Mim44 of the inner membrane import site seems to be an essential part of an ATP-dependent reaction cycle. However, the available results on the role played by ATP are controversial. Here we demonstrate that the mt-Hsp70.Mim44 complex contains ADP and that a nonhydrolyzable analog of ATP dissociates the mt-Hsp70.Mim44 complex in the presence of potassium ions. The previously reported requirement of ATP hydrolysis for complex dissociation was due to the use of a nonphysiological concentration of sodium ions. In the presence of potassium ions, mt-Hsp70 undergoes a conformational change that is not observed with a mutant Hsp70 defective in binding to Mim44. The mutant Hsp70 is able to bind substrate proteins, differentiating binding to Mim44 from binding to substrate proteins. We conclude that binding of ATP, not hydrolysis, is required to dissociate the mt-Hsp70.Mim44 complex and that the reaction cycle includes an ATP-induced conformational change of mt-Hsp70.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Difosfato de Adenosina/metabolismo , Adenilil Imidodifosfato/isolamento & purificação , Adenilil Imidodifosfato/metabolismo , Proteínas de Transporte/isolamento & purificação , Cromatografia de Afinidade , Ácido Edético/farmacologia , Proteínas de Choque Térmico HSP70/isolamento & purificação , Cinética , Magnésio/farmacologia , Proteínas de Membrana/isolamento & purificação , Proteínas do Complexo de Importação de Proteína Precursora MitocondrialRESUMO
Mitochondrial GrpE (Mge1p) is a mitochondrial cochaperone essential for viability of the yeast Saccharomyces cerevisiae. To study the role of Mge1p in the biogenesis of mitochondrial proteins, we isolated a conditional mutant allele of MGE1 which conferred a temperature-sensitive growth phenotype and led to the accumulation of mitochondrial preproteins after shifting of the cells to the restrictive temperature. The mutant Mge1 protein was impaired in its interaction with the matrix heat shock protein mt-Hsp70. The mutant mitochondria showed a delayed membrane translocation of preproteins, and the maturation of imported proteins was impaired, as evidenced by the retarded second proteolytic processing of a preprotein in the matrix. Moreover, the aggregation of imported proteins was decreased in the mutant mitochondria. The mutant Mge1p differentially modulated the interaction of mt-Hsp70 with preproteins compared with the wild type, resulting in decreased binding to preproteins in membrane transit and enhanced binding to fully imported proteins. We conclude that the interaction of Mge1p with mt-Hsp70 promotes the progress of the Hsp70 reaction cycle, which is essential for import and maturation of mitochondrial proteins.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico , Proteínas de Membrana Transportadoras , Mitocôndrias/metabolismo , Precursores de Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alelos , Sequência de Aminoácidos , Proteínas de Transporte/biossíntese , Proteínas de Transporte/isolamento & purificação , Sequência Conservada , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/isolamento & purificação , Genótipo , Proteínas de Choque Térmico HSP70/isolamento & purificação , Proteínas de Transporte da Membrana Mitocondrial , Chaperonas Moleculares , Dados de Sequência Molecular , Mutagênese Insercional , Fenótipo , Plasmídeos , Ligação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Homologia de Sequência de Aminoácidos , TemperaturaRESUMO
The 70 kDa heat shock proteins (Hsp70s) are ubiquitous molecular chaperones that are best known for their participation in protein folding. However, evidence is accumulating that Hsp70s perform several other cellular functions in cooperation with specific soluble or membrane-bound partner proteins. While the basic function of Hsp70s is explained by their ability to bind unfolded polypeptide segments, the partner proteins appear to customize them for specific roles such as involvement in protein traffic and folding, translocation of preproteins across membranes, and gene regulation.