MECANISMOS DE ADAPTACIÓN DE LA ACTIVIDAD MITOCONDRIAL EN RESPUESTA A ESTRÉS

[EN] Eukaryotic cells adapt to environmental changes ("stress") through signal transduction pathways which coordinate complex adaptive responses. Mitochondria are able to respond to different external stimuli in a dynamic manner. In previous studies, mitochondria were shown to play an important role in adaptation to hyperosmotic stress and defects in many mitochondrial functions cause sensitivity to this stress. In the present work, we investigate novel mechanisms of mitochondrial adaptation in response to stress. First of all, the role of the mitochondrial pyruvate carrier complex (MPC) in this adaptation was analyzed. This carrier is composed by three proteins in yeast: Mpc1, Mpc2 and Mpc3. MPC3 is upregulated upon salt stress and during a diauxic shift, which leads to an increase in Mpc3 protein abundance. HOG pathway, implicated in osmostress response, is needed for the efficient induction of MPC3 transcription. Our analysis suggests that amino acid biosynthesis, respiration rate and oxidative stress tolerance are regulated by changes in the Mpc protein composition of the mitochondria. In this way, Mpc2 is most abundant under fermentative non stress conditions and important for amino acid biosynthesis, while Mpc3 is the most abundant family member upon salt stress or when high respiration rates are required. In addition, Mpc3 stimulates respiration and enhances tolerance to oxidative stress. Therefore, our results identify that the regulated mitochondrial pyruvate uptake via different Mpc proteins might be an important determinant of respiration rate and stress resistance. Secondly, since pyruvate flux to mitochondria is modified according to environmental conditions, here we study also possible changes in electron transport chain complex subunits. We found that a switch to partially or completely respiratory energy sources causes selective degradation of respiratory complex I and III subunits. Moreover, this degradation was also observed when there was a specific organelle damage caused by valinomycin, to maintain cell homeostasis. Interestingly, the loss of Atg32 function only partially affected the respiratory complex specific degradation, while the Atg11 protein was absolutely required in this process. Fission and fusion machinery proteins (Fzo1 and Fis1) and some mitochondrial proteases (Yme1, Pim1 and Afg3) also have a role in the valinomycin-mediated mitophagy. This process might start by Atg11 accumulation in foci close to the mitochondria shortly after valinomycin treatment. In this work, we describe for the first time a specific mechanism of mitophagy mediated by damage in yeast, which opposes to the concept of a generalized degradation of the organelle.[ES] Las células eucariotas responden a cambios en su entorno ("estrés") a través de rutas de transmisión de señales que coordinan respuestas adaptativas muy complejas. Las mitocondrias son orgánulos muy dinámicos capaces de responder a diversos estímulos externos. En estudios anteriores, se demostró que la mitocondria tiene un papel en la adaptación a estrés hiperosmótico, ya que los mutantes con defectos en diversos componentes mitocondriales muestran mayor sensibilidad a este estrés. En este trabajo, se ha investigado nuevos mecanismos de adaptación de la actividad mitocondrial en respuesta a estrés. Por una parte, se ha estudiado el papel del complejo transportador de piruvato mitocondrial (MPC) en esta adaptación. Este transportador está conformado por tres proteínas en levadura: Mpc1, Mpc2 y Mpc3. El gen MPC3 sufre una fuerte inducción transcripcional en condiciones de estrés osmótico y cambio diáuxico, que se traduce en un aumento de la cantidad de proteína Mpc3. Esta regulación se vio que dependía de la ruta HOG, implicada en la respuesta a estrés osmótico, y no ocurría en Mpc1 y Mpc2. Se comprobó, además, que los cambios en la composición de MPC en la mitocondria regulaban la biosíntesis de aminoácidos, la capacidad respiratoria y la tolerancia a estrés oxidativo de la célula. De esta forma, Mpc2 es la proteína más abundante en condiciones fermentativas sin estrés y es necesaria para la biosíntesis de aminoácidos; mientras que Mpc3 es el miembro más abundante ante estrés salino o cuando se requiere una elevada tasa respiratoria. Además, Mpc3 estimula la respiración y aumenta la tolerancia a estrés oxidativo. Por tanto, nuestros resultados identifican que la entrada de piruvato en la mitocondria y su posterior uso están regulados por la composición específica de las subunidades del transportador y determina la tasa respiratoria y la resistencia a estrés. Por otra parte, dado que el flujo de piruvato a la mitocondria se modificaba en función de las condiciones ambientales, se quiso estudiar qué ocurría en los complejos de la cadena de transporte de electrones en estas condiciones. Se observó que los complejos I y III se degradaban ante elevadas tasas respiratorias, al parecer como un mecanismo de reciclaje. Además, ante un daño mitocondrial específico utilizando valinomicina, también existía una degradación específica de los complejos respiratorios I y III, para mantener la homeostasis celular. Este proceso es dependiente de Atg11, e independiente de Atg32. También parecen implicadas proteínas de la maquinaria de dinámica mitocondrial (Fzo1 y Fis1) y algunas proteasas mitocondriales (Yme1, Pim1 y Afg3). El inicio de este proceso parece producirse ante la aparición de foci de Atg11 cercanos a la mitocondria. Se describe por primera vez en levadura un mecanismo específico de mitofagia inducida por daño, que contrasta con el concepto de degradación generalizada del orgánulo.[CA] Les cèl·lules eucariotes responen a canvis al seu entorn ("estrès") a través de rutes de transmissió de senyals que coordinen respostes adaptatives molt complexes. Les mitocòndries són orgànuls molt dinàmics capaços de respondre a diversos estímuls externs. A estudis previs, es va demostrar que la mitocòndria té un paper en l'adaptació a estrès hiperosmòtic, ja què els mutants amb defectes en diversos components mitocondrials mostren major sensibilitat a aquest estrès. A aquest treball, s'ha analitzat nous mecanismes d'adaptació de l'activitat mitocondrial en resposta a estrès. Per una banda, s'ha estudiat el paper del complex transportador de piruvat mitocondrial (MPC) a aquesta adaptació. Aquest transportador està conformat per tres proteïnes en llevat: Mpc1, Mpc2 i Mpc3. El gen MPC3 pateix una forta inducció transcripcional en condicions d'estrès osmòtic i canvi diàuxic, que es tradueix en un augment de la quantitat de proteïna Mpc3. Aquesta regulació depèn de la ruta HOG, implicada en la resposta a estrès osmòtic, i no tenia lloc en Mpc1 i Mpc2. A més, es va comprovar que els canvis en la composició de MPC a la mitocòndria regulaven la biosíntesi de aminoàcids, la capacitat respiratòria i la tolerància a estrès oxidatiu de la cèl·lula. D'aquesta manera, Mpc2 és la proteïna més abundant en condicions fermentatives en absència d'estrès, mentre que Mpc3 és el membre més abundant davant d'estrès salí o quan és necessària una elevada taxa respiratòria. A més, Mpc3 estimula la respiració i augmenta la resistència a estrès oxidatiu. Per tant, els nostres resultats identifiquen que l'entrada de piruvat a la mitocòndria i el seu posterior ús estan regulats per la composició específica de les subunitats del transportador i determina la taxa respiratòria i la resistència a estrès. Per altra banda, com el flux de piruvat a la mitocòndria es modifica en funció de les condicions ambientals, es va voler estudiar què succeïa als complexes de la cadena de transport electrònic a aquestes condicions. Es va observar que els complexes I i III es degradaven davant d'elevades taxes respiratòries, com a mecanisme de reciclatge. A més, davant d'un dany mitocondrial específic utilitzant valinomicina, també existia una degradació específica dels complexes respiratoris I i III, per a mantenir l'homeòstasi cel·lular. Aquest procés és depenent d'Atg11, però independent d'Atg32. També semblen implicades proteïnes de la maquinària de dinàmica mitocondrial (Fzo1 i Fis1) i algunes proteases mitocondrials (Yme1, Afg3 i Pim1). L'inici d'aquest procés sembla produir-se per l'aparició de foci d'Atg11 propers a la mitocòndria. Per primera volta, es descriu en llevat un mecanisme específic de mitofagia induïda per dany, que contrasta amb el concepte de degradació generalitzada de l'orgànul.Timón Gómez, A. (2016). MECANISMOS DE ADAPTACIÓN DE LA ACTIVIDAD MITOCONDRIAL EN RESPUESTA A ESTRÉS [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/64873TESI

Differential regulation of mitochondrial pyruvate carrier genes modulates respiratory capacity and stress tolerance in yeast

Mpc proteins are highly conserved from yeast to humans and are necessary for the uptake of pyruvate at the inner mitochondrial membrane, which is used for leucine and valine biosynthesis and as a fuel for respiration. Our analysis of the yeast MPC gene family suggests that amino acid biosynthesis, respiration rate and oxidative stress tolerance are regulated by changes in the Mpc protein composition of the mitochondria. Mpc2 and Mpc3 are highly similar but functionally different: Mpc2 is most abundant under fermentative non stress conditions and important for amino acid biosynthesis, while Mpc3 is the most abundant family member upon salt stress or when high respiration rates are required. Accordingly, expression of the MPC3 gene is highly activated upon NaCl stress or during the transition from fermentation to respiration, both types of regulation depend on the Hog1 MAP kinase. Overexpression experiments show that gain of Mpc2 function leads to a severe respiration defect and ROS accumulation, while Mpc3 stimulates respiration and enhances tolerance to oxidative stress. Our results identify the regulated mitochondrial pyruvate uptake as an important determinant of respiration rate and stress resistance.This work was supported by Ministerio de Economia y Competitividad grant BFU2011-23326 to M.P.; A.T.-G. was supported by a JAE predoctoral grant from Consejo Superior de Investigaciones Cientificas. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Timón Gómez, A.; Proft ., MH.; Pascual-Ahuir Giner, MD. (2013). Differential regulation of mitochondrial pyruvate carrier genes modulates respiratory capacity and stress tolerance in yeast. PLoS ONE. 8(11):1-9. doi:10.1371/journal.pone.0079405S19811Murphy, M. P. (2008). How mitochondria produce reactive oxygen species. 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Ask yeast how to burn your fats: lessons learned from the metabolic adaptation to salt stress

[EN] Here, we review and update the recent advances in the metabolic control during the adaptive response of budding yeast to hyperosmotic and salt stress, which is one of the best understood signaling events at the molecular level. This environmental stress can be easily applied and hence has been exploited in the past to generate an impressively detailed and comprehensive model of cellular adaptation. It is clear now that this stress modulates a great number of different physiological functions of the cell, which altogether contribute to cellular survival and adaptation. Primary defense mechanisms are the massive induction of stress tolerance genes in the nucleus, the activation of cation transport at the plasma membrane, or the production and intracellular accumulation of osmolytes. At the same time and in a coordinated manner, the cell shuts down the expression of housekeeping genes, delays the progression of the cell cycle, inhibits genomic replication, and modulates translation efficiency to optimize the response and to avoid cellular damage. To this fascinating interplay of cellular functions directly regulated by the stress, we have to add yet another layer of control, which is physiologically relevant for stress tolerance. Salt stress induces an immediate metabolic readjustment, which includes the up-regulation of peroxisomal biomass and activity in a coordinated manner with the reinforcement of mitochondrial respiratory metabolism. Our recent findings are consistent with a model, where salt stress triggers a metabolic shift from fermentation to respiration fueled by the enhanced peroxisomal oxidation of fatty acids. We discuss here the regulatory details of this stress-induced metabolic shift and its possible roles in the context of the previously known adaptive functions.The work of the authors was supported by grants from Ministerio de Economía y Competitividad (BFU2011- 23326 and BFU2016-75792-R).Pascual-Ahuir Giner, MD.; Manzanares-Estreder, S.; Timón Gómez, A.; Proft ., MH. (2017). Ask yeast how to burn your fats: lessons learned from the metabolic adaptation to salt stress. Current Genetics. 64(1):63-69. https://doi.org/10.1007/s00294-017-0724-5S6369641Aguilera J, Prieto JA (2001) The Saccharomyces cerevisiae aldose reductase is implied in the metabolism of methylglyoxal in response to stress conditions. Curr Genet 39:273–283Albertyn J, Hohmann S, Thevelein JM, Prior BA (1994) GPD1, which encodes glycerol-3-phosphate dehydrogenase, is essential for growth under osmotic stress in Saccharomyces cerevisiae, and its expression is regulated by the high-osmolarity glycerol response pathway. 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Análisis de una familia de proteínas mitocondriales en levadura relacionadas con la proteína de cerebro humana Brp44

[ES] La levadura Saccharomyces cerevisiae contiene una familia de genes que codifican para proteínas ortólogas de la proteína de cerebro humana 44 (BRP44). Estas pequeñas proteínas mitocondriales están extraordinariamente conservadas a lo largo de la evolución desde las células de levadura a las células humanas. Esta familia proteica está codificada por los genes de levadura genes YGR243 (FMP43, SMOP1), YHR162W (SMOP2) y YGL080W (SMOP3). Se les ha renombrado como ¿SMOP¿ (Small Mitochondrial OsmoProtectant), debido a la fuerte inducción transcripcional del gen YGR243W mediante estrés hiperosmótico. En este trabajo se investiga cómo la ganancia y pérdida de la función de las proteínas Smop afecta a la tolerancia a estrés osmótico y oxidativo, a la producción de especies reactivas de oxígeno, y a la función y morfología mitocondrial. La morfología mitocondrial no se veía afectada en las cepas mutantes de deleción y de sobreexpresión de los genes SMOP. Además, se muestra que la deleción de genes SMOP individuales o de la familia entera no comprometía el crecimiento respiratorio o bajo condiciones severas de estrés osmótico. Sin embargo, las células ¿smop2 mostraban hipersensibilidad a estrés oxidativo. La sobreexpresión de los genes SMOP causaba la inhibición del crecimiento respiratorio y una disminución de la producción de ROS en un medio con fuentes de carbono no fermentables, pero no bajo condiciones de estrés oxidativo o salino. Estos datos preliminares sugieren un papel de las proteínas Smop en la regulación de la capacidad respiratoria y de la producción de ROS.[EN] The yeast Saccharomyces cerevisiae contains a family of genes encoding orthologs of the human brain protein 44 (BRP44). These small mitochondrial proteins are extraordinarily conserved through evolution from yeast to human cells. The protein family is encoded by the yeast genes YGR243 (FMP43, SMOP1), YHR162W (SMOP2) and YGL080W (SMOP3). They have been renamed ¿SMOP¿ for ¿Small Mitochondrial OsmoProtectant¿ because of the strong transcriptional induction of the YGR243W gene by hyperosmotic stress. Here we investigate how the loss and gain of function of Smop proteins affect osmotic and oxidative stress tolerance, the production of reactive oxygen species and the mitochondrial function and morphology. Mitochondrial morphology was not affected by the deletion or overexpression of the SMOP genes. We show that deletion of individual SMOP genes or of the entire family did not compromise respiratory growth or growth upon severe osmotic stress. However, ¿smop2 cells showed hypersensivity to oxidative stress. Overexpression of the SMOP genes caused inhibition of respiratory growth to different extent and reduced ROS production upon non fermentable carbon sources, but not upon oxidative or salt stress treatment. These preliminary data suggest a role for the Smop proteins in the regulation of respiratory capacity and ROS production.Timón Gómez, A. (2012). Análisis de una familia de proteínas mitocondriales en levadura relacionadas con la proteína de cerebro humana Brp44. http://hdl.handle.net/10251/17861Archivo delegad

Mitochondrial respiratory chain composition and organization in response to changing oxygen levels

Mitochondria are the major consumer of oxygen in eukaryotic cells, owing to the requirement of oxygen to generate ATP through the mitochondrial respiratory chain (MRC) and the oxidative phosphorylation system (OXPHOS). This aerobic energy transduction is more efficient than anaerobic processes such as glycolysis. Hypoxia, a condition in which environmental or intracellular oxygen levels are below the standard range, triggers an adaptive signaling pathway within the cell. When oxygen concentrations are low, hypoxia-inducible factors (HIFs) become stabilized and activated to mount a transcriptional response that triggers modulation of cellular metabolism to adjust to hypoxic conditions. Mitochondrial aerobic metabolism is one of the main targets of the hypoxic response to regulate its functioning and efficiency in the presence of decreased oxygen levels. During evolution, eukaryotic cells and tissues have increased the plasticity of their mitochondrial OXPHOS system to cope with metabolic needs in different oxygen contexts. In mammalian mitochondria, two factors contribute to this plasticity. First, several subunits of the multimeric MRC complexes I and IV exist in multiple tissue-specific and condition-specific isoforms. Second, the MRC enzymes can coexist organized as individual entities or forming supramolecular structures known as supercomplexes, perhaps in a dynamic manner to respond to environmental conditions and cellular metabolic demands. In this review, we will summarize the information currently available on oxygen-related changes in MRC composition and organization and will discuss gaps of knowledge and research opportunities in the field

HIGD-Driven Regulation of Cytochrome c Oxidase Biogenesis and Function

The biogenesis and function of eukaryotic cytochrome c oxidase or mitochondrial respiratory chain complex IV (CIV) undergo several levels of regulation to adapt to changing environmental conditions. Adaptation to hypoxia and oxidative stress involves CIV subunit isoform switch, changes in phosphorylation status, and modulation of CIV assembly and enzymatic activity by interacting factors. The latter include the Hypoxia Inducible Gene Domain (HIGD) family yeast respiratory supercomplex factors 1 and 2 (Rcf1 and Rcf2) and two mammalian homologs of Rcf1, the proteins HIGD1A and HIGD2A. Whereas Rcf1 and Rcf2 are expressed constitutively, expression of HIGD1A and HIGD2A is induced under stress conditions, such as hypoxia and/or low glucose levels. In both systems, the HIGD proteins localize in the mitochondrial inner membrane and play a role in the biogenesis of CIV as a free unit or as part as respiratory supercomplexes. Notably, they remain bound to assembled CIV and, by modulating its activity, regulate cellular respiration. Here, we will describe the current knowledge regarding the specific and overlapping roles of the several HIGD proteins in physiological and stress conditions

HIGD-Driven Regulation of Cytochrome c Oxidase Biogenesis and Function

The biogenesis and function of eukaryotic cytochrome oxidase or mitochondrial respiratory chain complex IV (CIV) undergo several levels of regulation to adapt to changing environmental conditions. Adaptation to hypoxia and oxidative stress involves CIV subunit isoform switch, changes in phosphorylation status, and modulation of CIV assembly and enzymatic activity by interacting factors. The latter include the Hypoxia Inducible Gene Domain (HIGD) family yeast respiratory supercomplex factors 1 and 2 (Rcf1 and Rcf2) and two mammalian homologs of Rcf1, the proteins HIGD1A and HIGD2A. Whereas Rcf1 and Rcf2 are expressed constitutively, expression of HIGD1A and HIGD2A is induced under stress conditions, such as hypoxia and/or low glucose levels. In both systems, the HIGD proteins localize in the mitochondrial inner membrane and play a role in the biogenesis of CIV as a free unit or as part as respiratory supercomplexes. Notably, they remain bound to assembled CIV and, by modulating its activity, regulate cellular respiration. Here, we will describe the current knowledge regarding the specific and overlapping roles of the several HIGD proteins in physiological and stress conditions

Effects of gain and loss of Mpc function on the oxygen consumption rate.

<p>The rate of oxygen consumption was measured in intact yeast cells corresponding to the indicated overexpression (A) or deletion strains (B). The control strain in the upper panel is wild type BY4741 with the empty overexpression vector and untransformed wild type in the lower panel. Data represent mean values +/− SD from at least three independent measurements. Mpc1 and Mpc2 overexpressing strains have significantly reduced oxygen consumption rates (p = 0.02), while Mpc3 overexpressing cells significantly increase the oxygen consumption rate (p = 0.05) according to the Students t-test (A). Oxygen consumption is significantly reduced (p = 0.05) in the <i>mpc1</i> and <i>mpc3</i> mutants as compared to wt according to the Students t-test (B).</p

The function of Mpc proteins in ROS balance and survival in stationary phase.

<p>A–B. The yeast strains overexpressing individual Mpc proteins described in Fig. 4 were grown in normal SD medium and subjected to a brief oxidative stress caused by hydrogen peroxide (A) or menadione (B). ROS was measured by the oxidation of dichlorodihydrofluorescein as described in “Experimental Procedures”. Fluorescence of the wild type before stress was arbitrarily set to 1. ROS levels were determined for three independent cultures in duplicate. Data presented are mean values +/− SD. Mpc1 and Mpc2 overexpressing strains have significantly increased ROS levels (p = 0.02 for panel A; p = 0.05 for panel B) as compared to wt according to the Students t-test (B). C. The survival in stationary phase in YPD medium of the indicated yeast strains was determined by plate assays after the indicated time.</p