RESUMO
A redox-regulated import pathway consisting of Mia40 and Erv1 mediates the import of cysteine-rich proteins into the mitochondrial intermembrane space. Mia40 is the oxidoreductase that inserts two disulfide bonds into the substrate simultaneously. However, Mia40 has one redox-active cysteine pair, resulting in ambiguity about how Mia40 accepts numerous electrons during substrate oxidation. In this study, we have addressed the oxidation of Tim13 in vitro and in organello. Reductants such as glutathione and ascorbate inhibited both the oxidation of the substrate Tim13 in vitro and the import of Tim13 and Cmc1 into isolated mitochondria. In addition, a ternary complex consisting of Erv1, Mia40, and substrate, linked by disulfide bonds, was not detected in vitro. Instead, Mia40 accepted six electrons from substrates, and this fully reduced Mia40 was sensitive to protease, indicative of conformational changes in the structure. Mia40 in mitochondria from the erv1-101 mutant was also trapped in a completely reduced state, demonstrating that Mia40 can accept up to six electrons as substrates are imported. Therefore, these studies support that Mia40 functions as an electron sink to facilitate the insertion of two disulfide bonds into substrates.
Assuntos
Elétrons , Regulação Fúngica da Expressão Gênica , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ácido Ascórbico/farmacologia , Dissulfetos/química , Dissulfetos/metabolismo , Glutationa/farmacologia , Metalochaperonas/genética , Metalochaperonas/metabolismo , Mitocôndrias/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/genética , Mutação , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Plasmídeos/química , Plasmídeos/metabolismo , Transporte Proteico , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Substâncias Redutoras/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de SinaisRESUMO
The endomembrane system of yeast contains different tail-anchored proteins that are post-translationally targeted to membranes via their C-terminal transmembrane domain. This hydrophobic segment could be hazardous in the cytosol if membrane insertion fails, resulting in the need for energy-dependent chaperoning and the degradation of aggregated tail-anchored proteins. A cascade of GET proteins cooperates in a conserved pathway to accept newly synthesized tail-anchored proteins from ribosomes and guide them to a receptor at the endoplasmic reticulum, where membrane integration takes place. It is, however, unclear how the GET system reacts to conditions of energy depletion that might prevent membrane insertion and hence lead to the accumulation of hydrophobic proteins in the cytosol. Here we show that the ATPase Get3, which accommodates the hydrophobic tail anchor of clients, has a dual function: promoting tail-anchored protein insertion when glucose is abundant and serving as an ATP-independent holdase chaperone during energy depletion. Like the generic chaperones Hsp42, Ssa2, Sis1 and Hsp104, we found that Get3 moves reversibly to deposition sites for protein aggregates, hence supporting the sequestration of tail-anchored proteins under conditions that prevent tail-anchored protein insertion. Our findings support a ubiquitous role for the cytosolic GET complex as a triaging platform involved in cellular proteostasis.
Assuntos
Adenosina Trifosfatases/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Modelos Moleculares , Transporte Proteico , Saccharomyces cerevisiae/metabolismoRESUMO
The Mia40 import pathway facilitates the import and oxidative folding of cysteine-rich protein substrates into the mitochondrial intermembrane space. Here we describe the in vitro and in organello oxidative folding of Cmc1, a twin CX(9)C-containing substrate, which contains an unpaired cysteine. In vitro, Cmc1 can be oxidized by the import receptor Mia40 alone when in excess or at a lower rate by only the sulfhydryl oxidase Erv1. However, physiological and efficient Cmc1 oxidation requires Erv1 and Mia40. Cmc1 forms a stable intermediate with Mia40 and is released from this interaction in the presence of Erv1. The three proteins are shown to form a ternary complex in mitochondria. Our results suggest that this mechanism facilitates efficient formation of multiple disulfides and prevents the formation of non-native disulfide bonds.
Assuntos
Metalochaperonas/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Motivos de Aminoácidos , Metalochaperonas/genética , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Ligação Proteica , Transporte Proteico/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
To understand and eventually predict the effects of changing redox conditions and oxidant levels on the physiology of an organism, it is essential to gain knowledge about its redoxome: the proteins whose activities are controlled by the oxidation status of their cysteine thiols. Here, we applied the quantitative redox proteomic method OxICAT to Saccharomyces cerevisiae and determined the in vivo thiol oxidation status of almost 300 different yeast proteins distributed among various cellular compartments. We found that a substantial number of cytosolic and mitochondrial proteins are partially oxidized during exponential growth. Our results suggest that prevailing redox conditions constantly control central cellular pathways by fine-tuning oxidation status and hence activity of these proteins. Treatment with sublethal H(2)O(2) concentrations caused a subset of 41 proteins to undergo substantial thiol modifications, thereby affecting a variety of different cellular pathways, many of which are directly or indirectly involved in increasing oxidative stress resistance. Classification of the identified protein thiols according to their steady-state oxidation levels and sensitivity to peroxide treatment revealed that redox sensitivity of protein thiols does not predict peroxide sensitivity. Our studies provide experimental evidence that the ability of protein thiols to react to changing peroxide levels is likely governed by both thermodynamic and kinetic parameters, making predicting thiol modifications challenging and de novo identification of peroxide sensitive protein thiols indispensable.
Assuntos
Peróxido de Hidrogênio/farmacologia , Oxidantes/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Oxirredução , Proteômica/métodosRESUMO
Tim54p, a component of the inner membrane TIM22 complex, does not directly mediate the import of inner membrane substrates but is required for assembly/stability of the 300-kD TIM22 complex. In addition, Deltatim54 yeast exhibit a petite-negative phenotype (also observed in yeast harboring mutations in the F1Fo ATPase, the ADP/ATP carrier, mitochondrial morphology components, or the i-AAA protease, Yme1p). Interestingly, other import mutants in our strain background are not petite-negative. We report that Tim54p is not involved in maintenance of mitochondrial DNA or mitochondrial morphology. Rather, Tim54p mediates assembly of an active Yme1p complex, after Yme1p is imported via the TIM23 pathway. Defective Yme1p assembly is likely the major contributing factor for the petite-negativity in strains lacking functional Tim54p. Thus, Tim54p has two independent functions: scaffolding/stability for the TIM22 membrane complex and assembly of Yme1p into a proteolytically active complex. As such, Tim54p links protein import, assembly, and turnover pathways in the mitochondrion.
Assuntos
Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteases Dependentes de ATP , Adenosina Trifosfatases/metabolismo , DNA Mitocondrial/metabolismo , Expressão Gênica , Glucose/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Viabilidade Microbiana , Proteínas de Transporte da Membrana Mitocondrial/deficiência , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Modelos Biológicos , Mutação/genética , Ligação Proteica , Transporte Proteico , Saccharomyces cerevisiae/citologiaRESUMO
Protein translocation pathways to the mitochondrial matrix and inner membrane have been well characterized. However, translocation into the intermembrane space, which was thought to be simply a modification of the traditional translocation pathways, is complex. The mechanism by which a subset of intermembrane space proteins, those with disulfide bonds, are translocated has been largely unknown until recently. Specifically, the intermembrane space proteins with disulfide bonds are imported via the mitochondrial intermembrane space assembly (MIA) pathway. Substrates are imported via a disulfide exchange relay with two components Mia40 and Erv1. This new breakthrough has resulted in novel concepts for assembly of proteins in the intermembrane space, suggesting that this compartment may be similar to that of the endoplasmic reticulum and the prokaryotic periplasm. As a better understanding of this pathway emerges, new paradigms for thiol-disulfide exchange mechanisms may be developed. Given that the intermembrane space is important for disease processes including apoptosis and neurodegeneration, new roles in regulation by oxidation-reduction chemistry seem likely to be relevant.
Assuntos
Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/química , Animais , Humanos , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Oxirredução , Dobramento de Proteína , Transporte Proteico/fisiologiaRESUMO
Multiple-choice assessments provide a straightforward way for instructors of large classes to collect data related to student understanding of key concepts at the beginning and end of a course. By tracking student performance over time, instructors receive formative feedback about their teaching and can assess the impact of instructional changes. The evidence of instructional effectiveness can in turn inform future instruction, and vice versa. In this study, we analyzed student responses on an optimized pretest and posttest administered during four different quarters in a large-enrollment biochemistry course. Student performance and the effect of instructional interventions related to three fundamental concepts-hydrogen bonding, bond energy, and pKa-were analyzed. After instructional interventions, a larger proportion of students demonstrated knowledge of these concepts compared with data collected before instructional interventions. Student responses trended from inconsistent to consistent and from incorrect to correct. The instructional effect was particularly remarkable for the later three quarters related to hydrogen bonding and bond energy. This study supports the use of multiple-choice instruments to assess the effectiveness of instructional interventions, especially in large classes, by providing instructors with quick and reliable feedback on student knowledge of each specific fundamental concept.
Assuntos
Bioquímica/educação , Compreensão , Feedback Formativo , Estudantes/psicologia , Ensino , Humanos , ConhecimentoRESUMO
The precise roles that oxidants play in lifespan and aging are still unknown. Here, we report the discovery that chronologically aging yeast cells undergo a sudden redox collapse, which affects over 80% of identified thiol-containing proteins. We present evidence that this redox collapse is not triggered by an increase in endogenous oxidants as would have been postulated by the free radical theory of aging. Instead it appears to be instigated by a substantial drop in cellular NADPH, which normally provides the electron source for maintaining cellular redox homeostasis. This decrease in NADPH levels occurs very early during lifespan and sets into motion a cascade that is predicted to down-regulate most cellular processes. Caloric restriction, a near-universal lifespan extending measure, increases NADPH levels and delays each facet of the cascade. Our studies reveal a time line of events leading up to the system-wide oxidation of the proteome days before cell death.DOI:http://dx.doi.org/10.7554/eLife.00306.001.
Assuntos
Mitose , NADP/metabolismo , Estresse Oxidativo , Proteoma , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Envelhecimento/metabolismo , Análise por Conglomerados , Regulação para Baixo , Homeostase , Oxirredução , Proteômica/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Tiorredoxina Dissulfeto Redutase/metabolismo , Fatores de TempoRESUMO
Mia40 and Erv1 execute a disulfide relay to import the small Tim proteins into the mitochondrial intermembrane space. Here, we have reconstituted the oxidative folding pathway in vitro with Tim13 as a substrate and determined the midpoint potentials of Mia40 and Tim13. Specifically, Mia40 served as a direct oxidant of Tim13, and Erv1 was required to reoxidize Mia40. During oxidation, four electrons were transferred from Tim13 with the insertion of two disulfide bonds in succession. The extent of Tim13 oxidation was directly dependent on Mia40 concentration and independent of Erv1 concentration. Characterization of the midpoint potentials showed that electrons flowed from Tim13 with a more negative midpoint potential of -310 mV via Mia40 with an intermediate midpoint potential of -290 mV to the C130-C133 pair of Erv1 with a positive midpoint potential of -150 mV. Intermediary complexes between Tim13-Mia40 and Mia40-Erv1 were trapped. Last, mutating C133 of the catalytic C130-C133 pair or C30 of the shuttle C30-C33 pair in Erv1 abolished oxidation of Tim13, whereas mutating the cysteines in the redox-active CPC motif, but not the structural disulfide linkages of the CX(9)C motif of Mia40, prevented Tim13 oxidation. Thus, we demonstrate that Mia40, Erv1, and oxygen are the minimal machinery for Tim13 oxidation.
Assuntos
Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas Mitocondriais/química , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Proteínas de Saccharomyces cerevisiae/química , Transdução de Sinais , Dicroísmo Circular , Dissulfetos/química , Dissulfetos/metabolismo , Eletroforese em Gel de Poliacrilamida , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Dobramento de Proteína , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Tolueno/análogos & derivados , Tolueno/química , Tolueno/metabolismoRESUMO
Previous studies of symmetry preferences in protein crystals suggest that symmetric proteins, such as homodimers, might crystallize more readily on average than asymmetric, monomeric proteins. Proteins that are naturally monomeric can be made homodimeric artificially by forming disulfide bonds between individual cysteine residues introduced by mutagenesis. Furthermore, by creating a variety of single-cysteine mutants, a series of distinct synthetic dimers can be generated for a given protein of interest, with each expected to gain advantage from its added symmetry and to exhibit a crystallization behavior distinct from the other constructs. This strategy was tested on phage T4 lysozyme, a protein whose crystallization as a monomer has been studied exhaustively. Experiments on three single-cysteine mutants, each prepared in dimeric form, yielded numerous novel crystal forms that cannot be realized by monomeric lysozyme. Six new crystal forms have been characterized. The results suggest that synthetic symmetrization may be a useful approach for enlarging the search space for crystallizing proteins.