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1.
Cell ; 180(2): 296-310.e18, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31978346

RESUMO

Mitochondria and lysosomes are functionally linked, and their interdependent decline is a hallmark of aging and disease. Despite the long-standing connection between these organelles, the function(s) of lysosomes required to sustain mitochondrial health remains unclear. Here, working in yeast, we show that the lysosome-like vacuole maintains mitochondrial respiration by spatially compartmentalizing amino acids. Defects in vacuole function result in a breakdown in intracellular amino acid homeostasis, which drives age-related mitochondrial decline. Among amino acids, we find that cysteine is most toxic for mitochondria and show that elevated non-vacuolar cysteine impairs mitochondrial respiration by limiting intracellular iron availability through an oxidant-based mechanism. Cysteine depletion or iron supplementation restores mitochondrial health in vacuole-impaired cells and prevents mitochondrial decline during aging. These results demonstrate that cysteine toxicity is a major driver of age-related mitochondrial deterioration and identify vacuolar amino acid compartmentation as a cellular strategy to minimize amino acid toxicity.


Assuntos
Cisteína/toxicidade , Ferro/metabolismo , Mitocôndrias/metabolismo , Aminoácidos/metabolismo , Senescência Celular/fisiologia , Cisteína/metabolismo , Homeostase , Lisossomos/metabolismo , Mitocôndrias/fisiologia , Mitofagia/fisiologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Vacúolos/metabolismo
2.
Cell ; 167(6): 1450-1452, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27912054

RESUMO

Structure determination by cryo-electron microscopy has approached atomic resolution and helped solve structures of large membrane-protein complexes that resisted crystallography. The 4.0 Å cryo-EM structure of one of the most intricate enzyme systems, the respirasome, in the mitochondrial inner membrane is reported in this issue of Cell.


Assuntos
Microscopia Crioeletrônica , Modelos Moleculares
3.
Mol Cell ; 71(4): 567-580.e4, 2018 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-30118679

RESUMO

The electron transport chain (ETC) is an important participant in cellular energy conversion, but its biogenesis presents the cell with numerous challenges. To address these complexities, the cell utilizes ETC assembly factors, which include the LYR protein family. Each member of this family interacts with the mitochondrial acyl carrier protein (ACP), the scaffold protein upon which the mitochondrial fatty acid synthesis (mtFAS) pathway builds fatty acyl chains from acetyl-CoA. We demonstrate that the acylated form of ACP is an acetyl-CoA-dependent allosteric activator of the LYR protein family used to stimulate ETC biogenesis. By tuning ETC assembly to the abundance of acetyl-CoA, which is the major fuel of the TCA cycle and ETC, this system could provide an elegant mechanism for coordinating the assembly of ETC complexes with one another and with substrate availability.


Assuntos
Acetilcoenzima A/metabolismo , Proteína de Transporte de Acila/metabolismo , Mitocôndrias/enzimologia , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/enzimologia , Proteína de Transporte de Acila/química , Proteína de Transporte de Acila/genética , Acilação , Regulação Alostérica , Sítios de Ligação , Ciclo do Ácido Cítrico/genética , Transporte de Elétrons/genética , Ácidos Graxos/biossíntese , Regulação Fúngica da Expressão Gênica , Mitocôndrias/genética , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Oxirredução , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
4.
Annu Rev Biochem ; 79: 537-62, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20205585

RESUMO

The current state of knowledge on how copper metallochaperones support the maturation of cuproproteins is reviewed. Copper is needed within mitochondria to supply the Cu(A) and intramembrane Cu(B) sites of cytochrome oxidase, within the trans-Golgi network to supply secreted cuproproteins and within the cytosol to supply superoxide dismutase 1 (Sod1). Subpopulations of copper-zinc superoxide dismutase also localize to mitochondria, the secretory system, the nucleus and, in plants, the chloroplast, which also requires copper for plastocyanin. Prokaryotic cuproproteins are found in the cell membrane and in the periplasm of gram-negative bacteria. Cu(I) and Cu(II) form tight complexes with organic molecules and drive redox chemistry, which unrestrained would be destructive. Copper metallochaperones assist copper in reaching vital destinations without inflicting damage or becoming trapped in adventitious binding sites. Copper ions are specifically released from copper metallochaperones upon contact with their cognate cuproproteins and metal transfer is thought to proceed by ligand substitution.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Cobre/metabolismo , Metalochaperonas/metabolismo , Bactérias/metabolismo , Proteínas de Transporte de Cátions/química , Citosol/metabolismo , Células Eucarióticas/metabolismo , Metalochaperonas/química , Mitocôndrias/metabolismo , Proteoma/análise
5.
J Biol Chem ; 294(8): 2815-2826, 2019 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-30593504

RESUMO

Zinc is an essential trace element that serves as a cofactor for enzymes in critical biochemical processes and also plays a structural role in numerous proteins. Zinc transporter ZIP4 (ZIP4) is a zinc importer required for dietary zinc uptake in the intestine and other cell types. Studies in cultured cells have reported that zinc stimulates the endocytosis of plasma membrane-localized ZIP4 protein, resulting in reduced cellular zinc uptake. Thus, zinc-regulated trafficking of ZIP4 is a key means for regulating cellular zinc homeostasis, but the underlying mechanisms are not well understood. In this study, we used mutational analysis, immunoblotting, HEK293 cells, and immunofluorescence microscopy to identify a histidine-containing motif (398HTH) in the first extracellular loop that is required for high sensitivity to low zinc concentrations in a zinc-induced endocytic response of mouse ZIP4 (mZIP4). Moreover, using synthetic peptides with selective substitutions and truncated mZIP4 variants, we provide evidence that histidine residues in this motif coordinate a zinc ion in mZIP4 homodimers at the plasma membrane. These findings suggest that 398HTH is an important zinc-sensing motif for eliciting high-affinity zinc-stimulated endocytosis of mZIP4 and provide insight into cellular mechanisms for regulating cellular zinc homeostasis in mammalian cells.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Endocitose/fisiologia , Matriz Extracelular/metabolismo , Histidina/química , Proteínas Mutantes/metabolismo , Mutação , Zinco/farmacologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Membrana Celular/metabolismo , Endocitose/efeitos dos fármacos , Células HEK293 , Histidina/metabolismo , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Transporte Proteico , Homologia de Sequência
6.
Proc Natl Acad Sci U S A ; 114(27): E5325-E5334, 2017 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-28634302

RESUMO

In eukaryotes, sulfur is mobilized for incorporation into multiple biosynthetic pathways by a cysteine desulfurase complex that consists of a catalytic subunit (NFS1), LYR protein (ISD11), and acyl carrier protein (ACP). This NFS1-ISD11-ACP (SDA) complex forms the core of the iron-sulfur (Fe-S) assembly complex and associates with assembly proteins ISCU2, frataxin (FXN), and ferredoxin to synthesize Fe-S clusters. Here we present crystallographic and electron microscopic structures of the SDA complex coupled to enzyme kinetic and cell-based studies to provide structure-function properties of a mitochondrial cysteine desulfurase. Unlike prokaryotic cysteine desulfurases, the SDA structure adopts an unexpected architecture in which a pair of ISD11 subunits form the dimeric core of the SDA complex, which clarifies the critical role of ISD11 in eukaryotic assemblies. The different quaternary structure results in an incompletely formed substrate channel and solvent-exposed pyridoxal 5'-phosphate cofactor and provides a rationale for the allosteric activator function of FXN in eukaryotic systems. The structure also reveals the 4'-phosphopantetheine-conjugated acyl-group of ACP occupies the hydrophobic core of ISD11, explaining the basis of ACP stabilization. The unexpected architecture for the SDA complex provides a framework for understanding interactions with acceptor proteins for sulfur-containing biosynthetic pathways, elucidating mechanistic details of eukaryotic Fe-S cluster biosynthesis, and clarifying how defects in Fe-S cluster assembly lead to diseases such as Friedreich's ataxia. Moreover, our results support a lock-and-key model in which LYR proteins associate with acyl-ACP as a mechanism for fatty acid biosynthesis to coordinate the expression, Fe-S cofactor maturation, and activity of the respiratory complexes.


Assuntos
Proteína de Transporte de Acila/metabolismo , Liases de Carbono-Enxofre/metabolismo , Proteínas Reguladoras de Ferro/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Sítios de Ligação , Liases de Carbono-Enxofre/química , Domínio Catalítico , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Proteínas de Ligação ao Ferro/química , Proteínas Reguladoras de Ferro/química , Cinética , Lipídeos/química , Mitocôndrias/metabolismo , Domínios Proteicos , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/metabolismo , Frataxina
7.
J Biol Chem ; 293(6): 1897-1898, 2018 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-29462794

RESUMO

A host of critical metalloproteins reside in mitochondria, where metallation occurs within the organelle after protein import. Although the pathways by which proteins are imported into the mitochondria are well known, the mechanisms by which their metal partners are imported are more obscure. A new study by Boulet et al. demonstrates that the mammalian SLC25A3 inner membrane transporter, previously known as a phosphate carrier, is also a functional Cu(I) importer, clarifying the source of mitochondrial copper and raising new questions about cellular copper homeostasis.


Assuntos
Cobre/metabolismo , Mitocôndrias/metabolismo , Animais , Transporte Biológico , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Camundongos , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas de Transporte de Fosfato/genética , Proteínas de Transporte de Fosfato/metabolismo
8.
J Biol Chem ; 293(15): 5585-5599, 2018 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-29475949

RESUMO

Cytochrome b (Cytb) is the only mitochondrial encoded subunit from the bc1 complex. Cbp3 and Cbp6 are chaperones necessary for translation of the COB mRNA and Cytb hemylation. Here we demonstrate that their role in translation is dispensable in some laboratory strains, whereas their role in Cytb hemylation seems to be universally conserved. BY4742 yeast requires Cbp3 and Cbp6 for efficient COB mRNA translation, whereas the D273-10b strain synthesizes Cytb at wildtype levels in the absence of Cbp3 and Cbp6. Steady-state levels of Cytb are close to wildtype in mutant D273-10b cells, and Cytb forms non-functional, supercomplex-like species with cytochrome c oxidase, in which at least core 1, cytochrome c1, and Rieske iron-sulfur subunits are present. We demonstrated that Cbp3 interacts with the mitochondrial ribosome and with the COB mRNA in both BY4742 and D273-10b strains. The polymorphism(s) causing the differential function of Cbp3, Cbp6, and the assembly feedback regulation of Cytb synthesis is of nuclear origin rather than mitochondrial, and Smt1, a COB mRNA-binding protein, does not seem to be involved in the observed differential phenotype. Our results indicate that the essential role of Cbp3 and Cbp6 is to assist Cytb hemylation and demonstrate that in the absence of heme b, Cytb can form non-functional supercomplexes with cytochrome c oxidase. Our observations support that an additional protein or proteins are involved in Cytb synthesis in some yeast strains.


Assuntos
Citocromos b/biossíntese , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/biossíntese , Chaperonas Moleculares/metabolismo , Biossíntese de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Citocromos b/genética , Citocromos c1/genética , Citocromos c1/metabolismo , Proteínas de Membrana/genética , Metiltransferases/genética , Metiltransferases/metabolismo , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Chaperonas Moleculares/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
9.
J Biol Chem ; 292(29): 12025-12040, 2017 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-28533431

RESUMO

Metallochaperones are a diverse family of trafficking molecules that provide metal ions to protein targets for use as cofactors. The copper chaperone for superoxide dismutase (Ccs1) activates immature copper-zinc superoxide dismutase (Sod1) by delivering copper and facilitating the oxidation of the Sod1 intramolecular disulfide bond. Here, we present structural, spectroscopic, and cell-based data supporting a novel copper-induced mechanism for Sod1 activation. Ccs1 binding exposes an electropositive cavity and proposed "entry site" for copper ion delivery on immature Sod1. Copper-mediated sulfenylation leads to a sulfenic acid intermediate that eventually resolves to form the Sod1 disulfide bond with concomitant release of copper into the Sod1 active site. Sod1 is the predominant disulfide bond-requiring enzyme in the cytoplasm, and this copper-induced mechanism of disulfide bond formation obviates the need for a thiol/disulfide oxidoreductase in that compartment.


Assuntos
Cobre/metabolismo , Cistina/metabolismo , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/metabolismo , Superóxido Dismutase/metabolismo , Substituição de Aminoácidos , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Cisteína/metabolismo , Ativação Enzimática , Estabilidade Enzimática , Humanos , Ligantes , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Mutagênese Sítio-Dirigida , Mutação , Oxirredução , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Superóxido Dismutase/química , Superóxido Dismutase/genética
10.
Crit Rev Biochem Mol Biol ; 50(2): 168-80, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25488574

RESUMO

Succinate dehydrogenase (or complex II; SDH) is a heterotetrameric protein complex that links the tribarboxylic acid cycle with the electron transport chain. SDH is composed of four nuclear-encoded subunits that must translocate independently to the mitochondria and assemble into a mature protein complex embedded in the inner mitochondrial membrane. Recently, it has become clear that failure to assemble functional SDH complexes can result in cancer and neurodegenerative syndromes. The effort to thoroughly elucidate the SDH assembly pathway has resulted in the discovery of four subunit-specific assembly factors that aid in the maturation of individual subunits and support the assembly of the intact complex. This review will focus on these assembly factors and assess the contribution of each factor to the assembly of SDH. Finally, we propose a model of the SDH assembly pathway that incorporates all extant data.


Assuntos
Mitocôndrias/enzimologia , Conformação Proteica , Subunidades Proteicas/química , Succinato Desidrogenase/química , Domínio Catalítico/genética , Núcleo Celular/enzimologia , Transporte de Elétrons/genética , Humanos , Mitocôndrias/química , Subunidades Proteicas/genética , Succinato Desidrogenase/classificação , Succinato Desidrogenase/genética
11.
J Mol Cell Cardiol ; 113: 22-32, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28962857

RESUMO

Calcium (Ca2+) influx into the mitochondrial matrix stimulates ATP synthesis. Here, we investigate whether mitochondrial Ca2+ transport pathways are altered in the setting of deficient mitochondrial energy synthesis, as increased matrix Ca2+ may provide a stimulatory boost. We focused on mitochondrial cardiomyopathies, which feature such dysfunction of oxidative phosphorylation. We study a mouse model where the main transcription factor for mitochondrial DNA (transcription factor A, mitochondrial, Tfam) has been disrupted selectively in cardiomyocytes. By the second postnatal week (10-15day old mice), these mice have developed a dilated cardiomyopathy associated with impaired oxidative phosphorylation. We find evidence of increased mitochondrial Ca2+ during this period using imaging, electrophysiology, and biochemistry. The mitochondrial Ca2+ uniporter, the main portal for Ca2+ entry, displays enhanced activity, whereas the mitochondrial sodium-calcium (Na+-Ca2+) exchanger, the main portal for Ca2+ efflux, is inhibited. These changes in activity reflect changes in protein expression of the corresponding transporter subunits. While decreased transcription of Nclx, the gene encoding the Na+-Ca2+ exchanger, explains diminished Na+-Ca2+ exchange, the mechanism for enhanced uniporter expression appears to be post-transcriptional. Notably, such changes allow cardiac mitochondria from Tfam knockout animals to be far more sensitive to Ca2+-induced increases in respiration. In the absence of Ca2+, oxygen consumption declines to less than half of control values in these animals, but rebounds to control levels when incubated with Ca2+. Thus, we demonstrate a phenotype of enhanced mitochondrial Ca2+ in a mitochondrial cardiomyopathy model, and show that such Ca2+ accumulation is capable of rescuing deficits in energy synthesis capacity in vitro.


Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Cardiomiopatias/metabolismo , Mitocôndrias Cardíacas/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , Fosforilação Oxidativa , Sódio/metabolismo , Trocador de Sódio e Cálcio/metabolismo
12.
J Biol Chem ; 291(33): 17417-26, 2016 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-27317660

RESUMO

The cellular transport of the cofactor heme and its biosynthetic intermediates such as protoporphyrin IX is a complex and highly coordinated process. To investigate the molecular details of this trafficking pathway, we created a synthetic lesion in the heme biosynthetic pathway by deleting the gene HEM15 encoding the enzyme ferrochelatase in S. cerevisiae and performed a genetic suppressor screen. Cells lacking Hem15 are respiratory-defective because of an inefficient heme delivery to the mitochondria. Thus, the biogenesis of mitochondrial cytochromes is negatively affected. The suppressor screen resulted in the isolation of respiratory-competent colonies containing two distinct missense mutations in Nce102, a protein that localizes to plasma membrane invaginations designated as eisosomes. The presence of the Nce102 mutant alleles enabled formation of the mitochondrial respiratory complexes and respiratory growth in hem15Δ cells cultured in supplemental hemin. Respiratory function in hem15Δ cells can also be restored by the presence of a heterologous plasma membrane heme permease (HRG-4), but the mode of suppression mediated by the Nce102 mutant is more efficient. Attenuation of the endocytic pathway through deletion of the gene END3 impaired the Nce102-mediated rescue, suggesting that the Nce102 mutants lead to suppression through the yeast endocytic pathway.


Assuntos
Endossomos/metabolismo , Heme/metabolismo , Mitocôndrias/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico Ativo/fisiologia , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Endossomos/genética , Ferroquelatase/genética , Ferroquelatase/metabolismo , Heme/genética , Mitocôndrias/genética , Mutação de Sentido Incorreto , Consumo de Oxigênio/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
13.
BMC Cancer ; 17(1): 497, 2017 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-28738844

RESUMO

BACKGROUND: Germline mutations in genes encoding subunits of succinate dehydrogenase (SDH) are associated with the development of pheochromocytoma (PC) and/or paraganglioma (PGL). As assembly factors have been identified as playing a role in maturation of individual SDH subunits and assembly of the functioning SDH complex, we hypothesized that SDHAF3 variants may be associated with PC/PGL and functionality of SDH. METHODS: DNA was extracted from the blood of 37 individuals (from 23 families) with germline SDH mutations and 18 PC/PGL (15 sporadic, 3 familial) and screened for mutations using a custom gene panel, containing SDHAF3 (SDH assembly factor 3) as well as eight known PC/PGL susceptibility genes. Molecular and functional consequences of an identified sequence variant of SDHAF3 were assessed in yeast and mammalian cells (HEK293). RESULTS: Using massively parallel sequencing, we identified a variant in SDHAF3, c.157 T > C (p.Phe53Leu), associated with increased prevalence in familial and sporadic PC/PGL (6.6%) when compared to normal populations (1.2% [1000 Genomes], p = 0.003; 2.1% [Exome Aggregation Consortium], p = 0.0063). In silico prediction tools suggest this variant is probably damaging to protein function, hence we assessed molecular and functional consequences of the resulting amino acid change (p.Phe53Leu) in yeast and human cells. We showed that introduction of SDHAF3 p.Phe53Leu into Sdh7 (ortholog of SDHAF3 in humans) null yeast resulted in impaired function, as observed by its failure to restore SDH activity when expressed in Sdh7 null yeast relative to WT SDHAF3. As SDHAF3 is involved in maturation of SDHB, we tested the functional impact of SDHAF3 c.157 T > C and various clinically relevant SDHB mutations on this interaction. Our in vitro studies in human cells show that SDHAF3 interacts with SDHB (residues 46 and 242), with impaired interaction observed in the presence of the SDHAF3 c.157 T > C variant. CONCLUSIONS: Our studies reveal novel insights into the biogenesis of SDH, uncovering a vital interaction between SDHAF3 and SDHB. We have shown that SDHAF3 interacts directly with SDHB (residue 242 being key to this interaction), and that a variant in SDHAF3 (c.157 T > C [p.Phe53Leu]) may be more prevalent in individuals with PC/PGL, and is hypomorphic via impaired interaction with SDHB.


Assuntos
Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Paraganglioma/enzimologia , Feocromocitoma/enzimologia , Succinato Desidrogenase/metabolismo , Animais , Feminino , Células HEK293 , Humanos , Masculino , Simulação de Acoplamento Molecular , Paraganglioma/genética , Feocromocitoma/genética , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Saccharomyces cerevisiae , Análise de Sequência de DNA , Succinato Desidrogenase/genética , Sus scrofa/metabolismo
14.
J Biol Chem ; 289(9): 6133-41, 2014 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-24421313

RESUMO

Yeast cells deficient in the Rieske iron-sulfur subunit (Rip1) of ubiquinol-cytochrome c reductase (bc1) accumulate a late core assembly intermediate, which weakly associates with cytochrome oxidase (CcO) in a respiratory supercomplex. Expression of the N-terminal half of Rip1, which lacks the C-terminal FeS-containing globular domain (designated N-Rip1), results in a marked stabilization of trimeric and tetrameric bc1-CcO supercomplexes. Another bc1 mutant (qcr9Δ) stalled at the same assembly intermediate is likewise converted to stable supercomplex species by the expression of N-Rip1, but not by expression of intact Rip1. The N-Rip1-induced stabilization of bc1-CcO supercomplexes is independent of the Bcs1 translocase, which mediates Rip1 translocation during bc1 biogenesis. N-Rip1 induces the stabilization of bc1-CcO supercomplexes through an enhanced formation of CcO. The association of N-Rip1 with the late core bc1 assembly intermediate appears to confer stabilization of a CcO assembly intermediate. This induced stabilization of CcO is dependent on the Rcf1 supercomplex stabilization factor and only partially dependent on the presence of cardiolipin. N-Rip1 exerts a related induction of CcO stabilization in WT yeast, resulting in enhanced respiration. Additionally, the impact of CcO stabilization on supercomplexes was observed by means other than expression of N-Rip1 (via overexpression of CcO subunits Cox4 and Cox5a), demonstrating that this is a general phenomenon. This study presents the first evidence showing that supercomplexes can be stabilized by the stimulated formation of CcO.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Transporte de Elétrons/fisiologia , Complexo IV da Cadeia de Transporte de Elétrons/genética , Estabilidade Enzimática/fisiologia , Mutação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
15.
J Biol Chem ; 288(33): 23884-92, 2013 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-23846699

RESUMO

Saccharomyces cerevisiae must import copper into the mitochondrial matrix for eventual assembly of cytochrome c oxidase. This copper is bound to an anionic fluorescent molecule known as the copper ligand (CuL). Here, we identify for the first time a mitochondrial carrier family protein capable of importing copper into the matrix. In vitro transport of the CuL into the mitochondrial matrix was saturable and temperature-dependent. Strains with a deletion of PIC2 grew poorly on copper-deficient non-fermentable medium supplemented with silver and under respiratory conditions when challenged with a matrix-targeted copper competitor. Mitochondria from pic2Δ cells had lower total mitochondrial copper and exhibited a decreased capacity for copper uptake. Heterologous expression of Pic2 in Lactococcus lactis significantly enhanced CuL transport into these cells. Therefore, we propose a novel role for Pic2 in copper import into mitochondria.


Assuntos
Cobre/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Transporte de Fosfato/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico , Deleção de Genes , Humanos , Lactococcus lactis/metabolismo , Ligantes , Fenótipo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Prata/metabolismo , Superóxido Dismutase/metabolismo
16.
J Biol Chem ; 288(3): 1696-705, 2013 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-23192348

RESUMO

Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes, where it is synthesized locally and plays a critical role in mitochondrial bioenergetic functions. The importance of CL in human health is underscored by the observation that perturbation of CL biosynthesis causes the severe genetic disorder Barth syndrome. To fully understand the cellular response to the loss of CL, we carried out genome-wide expression profiling of the yeast CL mutant crd1Δ. Our results show that the loss of CL in this mutant leads to increased expression of iron uptake genes accompanied by elevated levels of mitochondrial iron and increased sensitivity to iron and hydrogen peroxide. Previous studies have shown that increased mitochondrial iron levels result from perturbations in iron-sulfur (Fe-S) cluster biogenesis. Consistent with an Fe-S defect, deletion of ISU1, one of two ISU genes that encode the mitochondrial Fe-S scaffolding protein essential for the synthesis of Fe-S clusters, led to synthetic growth defects with the crd1Δ mutant. We further show that crd1Δ cells have reduced activities of mitochondrial Fe-S enzymes (aconitase, succinate dehydrogenase, and ubiquinol-cytochrome c oxidoreductase), as well as cytosolic Fe-S enzymes (sulfite reductase and isopropylmalate isomerase). Increased expression of ATM1 or YAP1 did not rescue the Fe-S defects in crd1Δ. These findings show for the first time that CL is required for Fe-S biogenesis to maintain mitochondrial and cellular iron homeostasis.


Assuntos
Cardiolipinas/metabolismo , Regulação Fúngica da Expressão Gênica , Ferro/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Saccharomyces cerevisiae/genética , Aconitato Hidratase/genética , Aconitato Hidratase/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Perfilação da Expressão Gênica , Humanos , Peróxido de Hidrogênio/metabolismo , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Isomerases/genética , Isomerases/metabolismo , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mutação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismo , Sulfito Redutase (NADPH)/genética , Sulfito Redutase (NADPH)/metabolismo
17.
Biochim Biophys Acta ; 1827(5): 627-36, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23380393

RESUMO

The Succinate Dehydrogenase (SDH) heterotetrameric complex catalyzes the oxidation of succinate to fumarate in the tricarboxylic acid (TCA) cycle and in the aerobic respiratory chains of eukaryotes and bacteria. Essential in this catalysis is the covalently-linked cofactor flavin adenine dinucleotide (FAD) in subunit1 (Sdh1) of the SDH enzyme complex. The mechanism of FAD insertion and covalent attachment to Sdh1 is unknown. Our working concept of this flavinylation process has relied mostly on foundational works from the 1990s and by applying the principles learned from other enzymes containing a similarly linked FAD. The discovery of the flavinylation factor Sdh5, however, has provided new insight into the possible mechanism associated with Sdh1 flavinylation. This review focuses on encapsulating prior and recent advances towards understanding the mechanism associated with flavinylation of Sdh1 and how this flavinylation process affects the overall assembly of SDH. This article is part of a Special Issue entitled: Respiratory complex II: Role in cellular physiology and disease.


Assuntos
Complexo II de Transporte de Elétrons/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Proteínas Mitocondriais/metabolismo , Succinato Desidrogenase/metabolismo , Complexo II de Transporte de Elétrons/química , Humanos , Proteínas Mitocondriais/química , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Succinato Desidrogenase/química
18.
Biochim Biophys Acta ; 1827(3): 285-93, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23168492

RESUMO

The mammalian Complex III (CIII) assembly process is yet to be completely understood. There is still a lack in understanding of how the structural subunits are put together and which additional factors are involved. Here we describe the identification and characterization of LYRM7, a human protein displaying high sequence homology to the Saccharomyces cerevisiae protein Mzm1, which was recently shown as an assembly factor for Rieske Fe-S protein incorporation into the yeast cytochrome bc(1) complex. We conclude that human LYRM7, which we propose to be renamed MZM1L (MZM1-like), works as a human Rieske Fe-S protein (UQCRFS1) chaperone, binding to this subunit within the mitochondrial matrix and stabilizing it prior to its translocation and insertion into the late CIII dimeric intermediate within the mitochondrial inner membrane. Thus, LYRM7/MZM1L is a novel human CIII assembly factor involved in the UQCRFS1 insertion step, which enables formation of the mature and functional CIII enzyme.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Proteínas Mitocondriais/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Sequência de Aminoácidos , Animais , Complexo III da Cadeia de Transporte de Elétrons/fisiologia , Células HEK293 , Células HeLa , Humanos , Camundongos , Proteínas Mitocondriais/fisiologia , Chaperonas Moleculares/fisiologia , Dados de Sequência Molecular
19.
Proc Natl Acad Sci U S A ; 108(7): 2729-34, 2011 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-21282621

RESUMO

Hydrogen peroxide is thought to regulate cellular processes by direct oxidation of numerous cellular proteins, whereas antioxidants, most notably thiol peroxidases, are thought to reduce peroxides and inhibit H(2)O(2) response. However, thiol peroxidases have also been implicated in activation of transcription factors and signaling. It remains unclear if these enzymes stimulate or inhibit redox regulation and whether this regulation is widespread or limited to a few cellular components. Herein, we found that Saccharomyces cerevisiae cells lacking all eight thiol peroxidases were viable and withstood redox stresses. They transcriptionally responded to various redox treatments, but were unable to activate and repress gene expression in response to H(2)O(2). Further studies involving redox transcription factors suggested that thiol peroxidases are major regulators of global gene expression in response to H(2)O(2). The data suggest that thiol peroxidases sense and transfer oxidative signals to the signaling proteins and regulate transcription, whereas a direct interaction between H(2)O(2) and other cellular proteins plays a secondary role.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Peróxido de Hidrogênio/toxicidade , Peroxidases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Transdução de Sinais/efeitos dos fármacos , Sequência de Bases , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo/genética , Peroxidases/deficiência , Fenótipo , Proteínas Ribossômicas/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Análise de Sequência de DNA , Transdução de Sinais/fisiologia
20.
Elife ; 132024 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-38251707

RESUMO

Mitochondrial membrane potential directly powers many critical functions of mitochondria, including ATP production, mitochondrial protein import, and metabolite transport. Its loss is a cardinal feature of aging and mitochondrial diseases, and cells closely monitor membrane potential as an indicator of mitochondrial health. Given its central importance, it is logical that cells would modulate mitochondrial membrane potential in response to demand and environmental cues, but there has been little exploration of this question. We report that loss of the Sit4 protein phosphatase in yeast increases mitochondrial membrane potential, both by inducing the electron transport chain and the phosphate starvation response. Indeed, a similarly elevated mitochondrial membrane potential is also elicited simply by phosphate starvation or by abrogation of the Pho85-dependent phosphate sensing pathway. This enhanced membrane potential is primarily driven by an unexpected activity of the ADP/ATP carrier. We also demonstrate that this connection between phosphate limitation and enhancement of mitochondrial membrane potential is observed in primary and immortalized mammalian cells as well as in Drosophila. These data suggest that mitochondrial membrane potential is subject to environmental stimuli and intracellular signaling regulation and raise the possibility for therapeutic enhancement of mitochondrial function even in defective mitochondria.


Assuntos
Fosfatos , Saccharomyces cerevisiae , Animais , Potencial da Membrana Mitocondrial , Fosfatos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo , Respiração , Mamíferos/metabolismo
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