RESUMEN
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.
Asunto(s)
Fosfatos , Saccharomyces cerevisiae , Animales , Potencial de la Membrana Mitocondrial , Fosfatos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfato/metabolismo , Respiración , Mamíferos/metabolismoRESUMEN
Synthesis of iron-sulfur (Fe/S) clusters in living cells requires scaffold proteins for both facile synthesis and subsequent transfer of clusters to target apoproteins. The human mitochondrial ISCU2 scaffold protein is part of the core ISC (iron-sulfur cluster assembly) complex that synthesizes a bridging [2Fe-2S] cluster on dimeric ISCU2. Initial iron and sulfur loading onto monomeric ISCU2 have been elucidated biochemically, yet subsequent [2Fe-2S] cluster formation and dimerization of ISCU2 is mechanistically ill-defined. Our structural, biochemical and cell biological experiments now identify a crucial function of the universally conserved N-terminal Tyr35 of ISCU2 for these late reactions. Mixing two, per se non-functional ISCU2 mutant proteins with oppositely charged Asp35 and Lys35 residues, both bound to different cysteine desulfurase complexes NFS1-ISD11-ACP, restores wild-type ISCU2 maturation demonstrating that ionic forces can replace native Tyr-Tyr interactions during dimerization-induced [2Fe-2S] cluster formation. Our studies define the essential mechanistic role of Tyr35 in the reaction cycle of de novo mitochondrial [2Fe-2S] cluster synthesis.
Asunto(s)
Dimerización , Proteínas Hierro-Azufre/química , Tirosina/química , Apoproteínas , Liasas de Carbono-Azufre , Cristalografía por Rayos X , Ferredoxinas , Células HeLa , Humanos , Hierro , Mitocondrias , Proteínas Mutantes , Proteínas Recombinantes , AzufreRESUMEN
Cells harbor two systems for fatty acid synthesis, one in the cytoplasm (catalyzed by fatty acid synthase, FASN) and one in the mitochondria (mtFAS). In contrast to FASN, mtFAS is poorly characterized, especially in higher eukaryotes, with the major product(s), metabolic roles, and cellular function(s) being essentially unknown. Here we show that hypomorphic mtFAS mutant mouse skeletal myoblast cell lines display a severe loss of electron transport chain (ETC) complexes and exhibit compensatory metabolic activities including reductive carboxylation. This effect on ETC complexes appears to be independent of protein lipoylation, the best characterized function of mtFAS, as mutants lacking lipoylation have an intact ETC. Finally, mtFAS impairment blocks the differentiation of skeletal myoblasts in vitro. Together, these data suggest that ETC activity in mammals is profoundly controlled by mtFAS function, thereby connecting anabolic fatty acid synthesis with the oxidation of carbon fuels.
In human, plant and other eukaryotic cells, fats are an important source of energy and also play many other roles including waterproofing, thermal insulation and energy storage. Eukaryotic cells have two systems that make the building blocks of fats (known as fatty acids) and one of these systems, called the mtFAS pathway, operates in small compartments known as mitochondria. This pathway only has one known product, a small fat molecule called lipoic acid, which mitochondria attach to several enzymes to allow them to work properly. The main role of mitochondria is to break down fats and other molecules to release chemical energy that powers many processes in cells. They achieve this using large groups of proteins known as ETC complexes. To build these complexes, families of proteins known as ETC assembly factors carefully coordinate the assembly of many proteins and small molecules into specific structures. However, it remains unclear precisely how this process works. Here, Nowinski et al. used a gene editing technique to mutate the genes encoding three enzymes in the mtFAS pathway in mammalian cells. The experiments found that the mutant cells had fewer ETC complexes and seemed to be less able to break down fats and other molecules than 'normal' cells. Furthermore, a family of ETC assembly factors were less stable in the mutant cells. These findings suggest that the mtFAS pathway controls how mitochondria assemble ETC complexes. Further experiments indicated that lipoic acid is not involved in the assembly of ETC complexes and that the mtFAS pathway produces another, as yet unidentified, product that regulates this process, instead. MEPAN syndrome is a rare neurological disorder that leads to progressive loss of control of movement, slurred speech and impaired vision in children. Patients with this syndrome have genetic mutations affecting components of the mtFAS pathway, therefore, a better understanding of how the pathway works may help researchers develop new treatments in the future. More broadly, these findings will have important ramifications for many other situations in which the activity of ETC complexes in mitochondria is modified.
Asunto(s)
Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Ácidos Grasos/biosíntesis , Mitocondrias/metabolismo , Mioblastos/fisiología , Animales , Diferenciación Celular , Línea Celular , Proteínas del Complejo de Cadena de Transporte de Electrón/genética , Células HEK293 , Humanos , Lipoilación/genética , Ratones , Oxidación-ReducciónRESUMEN
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.
Asunto(s)
Cisteína/toxicidad , Hierro/metabolismo , Mitocondrias/metabolismo , Aminoácidos/metabolismo , Senescencia Celular/fisiología , Cisteína/metabolismo , Homeostasis , Lisosomas/metabolismo , Mitocondrias/fisiología , Mitofagia/fisiología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Vacuolas/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Transporte de Catión/metabolismo , Endocitosis/fisiología , Matriz Extracelular/metabolismo , Histidina/química , Proteínas Mutantes/metabolismo , Mutación , Zinc/farmacología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas de Transporte de Catión/química , Proteínas de Transporte de Catión/genética , Membrana Celular/metabolismo , Endocitosis/efectos de los fármacos , Células HEK293 , Histidina/metabolismo , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Transporte de Proteínas , Homología de SecuenciaRESUMEN
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.
Asunto(s)
Acetilcoenzima A/metabolismo , Proteína Transportadora de Acilo/metabolismo , Mitocondrias/enzimología , Procesamiento Proteico-Postraduccional , Saccharomyces cerevisiae/enzimología , Proteína Transportadora de Acilo/química , Proteína Transportadora de Acilo/genética , Acilación , Regulación Alostérica , Sitios de Unión , Ciclo del Ácido Cítrico/genética , Transporte de Electrón/genética , Ácidos Grasos/biosíntesis , Regulación Fúngica de la Expresión Génica , Mitocondrias/genética , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Modelos Moleculares , Oxidación-Reducción , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de 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/metabolismoRESUMEN
In this issue of Cell Chemical Biology, Pandey et al. (2018) identified that mitochondrial cysteine desulfurase provides the sulfur species used for tRNALys, tRNAGlu, and tRNAGln thiouridine modification in the cytoplasm. A low-mass sulfur species is exported by the mitochondrial Atm1 transporter and utilized in the thio-modifications.
Asunto(s)
Mitocondrias , ARN de Transferencia , Citosol , Azufre , TiouridinaRESUMEN
Iron-sulfur clusters (Fe/S clusters) are essential cofactors required throughout the clades of biology for performing a myriad of unique functions including nitrogen fixation, ribosome assembly, DNA repair, mitochondrial respiration, and metabolite catabolism. Although Fe/S clusters can be synthesized in vitro and transferred to a client protein without enzymatic assistance, biology has evolved intricate mechanisms to assemble and transfer Fe/S clusters within the cellular environment. In eukaryotes, the foundation of all cellular clusters starts within the mitochondria. The focus of this review is to detail the mitochondrial Fe/S biogenesis (ISC) pathway along with the Fe/S cluster transfer steps necessary to mature Fe/S proteins. New advances in our understanding of the mitochondrial Fe/S biogenesis machinery will be highlighted. Additionally, we will address various experimental approaches that have been successful in the identification and characterization of components of the ISC pathway.
Asunto(s)
Vías Biosintéticas , Proteínas Hierro-Azufre/metabolismo , Proteínas Mitocondriales/metabolismo , Proteína Transportadora de Acilo/metabolismo , Animales , Proteínas Portadoras/metabolismo , Humanos , Mitocondrias/metabolismoRESUMEN
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.
Asunto(s)
Cobre/metabolismo , Mitocondrias/metabolismo , Animales , Transporte Biológico , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Humanos , Ratones , Mitocondrias/genética , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Proteínas de Transporte de Fosfato/genética , Proteínas de Transporte de Fosfato/metabolismoRESUMEN
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.
Asunto(s)
Citocromos b/biosíntesis , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/biosíntesis , Chaperonas Moleculares/metabolismo , Biosíntesis de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Citocromos b/genética , Citocromos c1/genética , Citocromos c1/metabolismo , Proteínas de la Membrana/genética , Metiltransferasas/genética , Metiltransferasas/metabolismo , Mitocondrias/genética , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Chaperonas Moleculares/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
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.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , Cardiomiopatías/metabolismo , Mitocondrias Cardíacas/metabolismo , Animales , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/metabolismo , Fosforilación Oxidativa , Sodio/metabolismo , Intercambiador de Sodio-Calcio/metabolismoRESUMEN
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.
Asunto(s)
Predisposición Genética a la Enfermedad , Mutación de Línea Germinal , Paraganglioma/enzimología , Feocromocitoma/enzimología , Succinato Deshidrogenasa/metabolismo , Animales , Femenino , Células HEK293 , Humanos , Masculino , Simulación del Acoplamiento Molecular , Paraganglioma/genética , Feocromocitoma/genética , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Saccharomyces cerevisiae , Análisis de Secuencia de ADN , Succinato Deshidrogenasa/genética , Sus scrofa/metabolismoRESUMEN
Copper zinc superoxide dismutase (Sod1) is a critical enzyme in limiting reactive oxygen species in both the cytosol and the mitochondrial intermembrane space. Sod1 dismutes superoxide anions to hydrogen peroxide and oxygen. The catalytic reaction is dependent on an active site copper ion and a disulfide bonded conformation. The activation of Sod1 is mediated by its chaperone Ccs1. The mechanism of Ccs1-mediated Sod1 activation involves both insertion of the catalytic copper ion and mediating disulfide bond formation. Since Sod1 is a highly abundant enzyme residing within the highly reducing cytoplasm, the question of disulfide bond formation is significant yet unresolved. The processes involved in Sod1 activation are reviewed with a focus on copper ion insertion and disulfide bond formation.
Asunto(s)
Cobre/metabolismo , Disulfuros/metabolismo , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa-1/metabolismo , Zinc/metabolismo , Animales , Activación Enzimática , Humanos , Oxidación-ReducciónRESUMEN
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.
Asunto(s)
Proteína Transportadora de Acilo/metabolismo , Liasas de Carbono-Azufre/metabolismo , Proteínas Reguladoras del Hierro/metabolismo , Proteínas Hierro-Azufre/metabolismo , Sitios de Unión , Liasas de Carbono-Azufre/química , Dominio Catalítico , Cromatografía de Gases y Espectrometría de Masas , Humanos , Proteínas de Unión a Hierro/química , Proteínas Reguladoras del Hierro/química , Cinética , Lípidos/química , Mitocondrias/metabolismo , Dominios Proteicos , Estructura Secundaria de Proteína , Saccharomyces cerevisiae/metabolismo , FrataxinaRESUMEN
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.
Asunto(s)
Cobre/metabolismo , Cistina/metabolismo , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Saccharomyces cerevisiae/metabolismo , Superóxido Dismutasa/metabolismo , Sustitución de Aminoácidos , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Cisteína/metabolismo , Activación Enzimática , Estabilidad de Enzimas , Humanos , Ligandos , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Mutagénesis Sitio-Dirigida , Mutación , Oxidación-Reducción , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Superóxido Dismutasa/química , Superóxido Dismutasa/genéticaRESUMEN
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.
Asunto(s)
Microscopía por Crioelectrón , Modelos MolecularesRESUMEN
Mitochondrial fatty acid synthesis (FASII) and iron sulfur cluster (FeS) biogenesis are both vital biosynthetic processes within mitochondria. In this study, we demonstrate that the mitochondrial acyl carrier protein (ACP), which has a well-known role in FASII, plays an unexpected and evolutionarily conserved role in FeS biogenesis. ACP is a stable and essential subunit of the eukaryotic FeS biogenesis complex. In the absence of ACP, the complex is destabilized resulting in a profound depletion of FeS throughout the cell. This role of ACP depends upon its covalently bound 4'-phosphopantetheine (4-PP)-conjugated acyl chain to support maximal cysteine desulfurase activity. Thus, it is likely that ACP is not simply an obligate subunit but also exploits the 4-PP-conjugated acyl chain to coordinate mitochondrial fatty acid and FeS biogenesis.
Asunto(s)
Proteína Transportadora de Acilo/metabolismo , Ácidos Grasos/metabolismo , Compuestos de Hierro/metabolismo , Mitocondrias/metabolismo , Compuestos de Azufre/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
Iron-sulfur (Fe-S) clusters are essential for many cellular processes, ranging from aerobic respiration, metabolite biosynthesis, ribosome assembly and DNA repair. Mutations in NFU1 and BOLA3 have been linked to genetic diseases with defects in mitochondrial Fe-S centers. Through genetic studies in yeast, we demonstrate that Nfu1 functions in a late step of [4Fe-4S] cluster biogenesis that is of heightened importance during oxidative metabolism. Proteomic studies revealed Nfu1 physical interacts with components of the ISA [4Fe-4S] assembly complex and client proteins that need [4Fe-4S] clusters to function. Additional studies focused on the mitochondrial BolA proteins, Bol1 and Bol3 (yeast homolog to human BOLA3), revealing that Bol1 functions earlier in Fe-S biogenesis with the monothiol glutaredoxin, Grx5, and Bol3 functions late with Nfu1. Given these observations, we propose that Nfu1, assisted by Bol3, functions to facilitate Fe-S transfer from the biosynthetic apparatus to the client proteins preventing oxidative damage to [4Fe-4S] clusters.
Asunto(s)
Proteínas Hierro-Azufre/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico , Proteínas Mitocondriales/genética , Proteoma/análisis , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
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.