RESUMEN
At the pyruvate branch point, the fermentative and oxidative metabolic routes diverge. Pyruvate can be transformed either into lactate in mammalian cells or into ethanol in yeast, or transported into mitochondria to fuel ATP production by oxidative phosphorylation. The recently discovered mitochondrial pyruvate carrier (MPC), encoded by MPC1, MPC2, and MPC3 in yeast, is required for uptake of pyruvate into the organelle. Here, we show that while expression of Mpc1 is not dependent on the carbon source, expression of Mpc2 and Mpc3 is specific to fermentative or respiratory conditions, respectively. This gives rise to two alternative carrier complexes that we have termed MPCFERM and MPCOX. By constitutively expressing the two alternative complexes in yeast deleted for all three endogenous genes, we show that MPCOX has a higher transport activity than MPCFERM, which is dependent on the C-terminus of Mpc3. We propose that the alternative MPC subunit expression in yeast provides a way of adapting cellular metabolism to the nutrient availability.
Asunto(s)
Proteínas de Transporte de Anión/biosíntesis , Proteínas de la Membrana/biosíntesis , Mitocondrias/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/biosíntesis , Ácido Pirúvico/metabolismo , Proteínas de Saccharomyces cerevisiae/biosíntesis , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte de Anión/genética , Transporte Biológico Activo/fisiología , Regulación Fúngica de la Expresión Génica/fisiología , Proteínas de la Membrana/genética , Mitocondrias/genética , Proteínas de Transporte de Membrana Mitocondrial , Complejos Multiproteicos/biosíntesis , Complejos Multiproteicos/genética , Consumo de Oxígeno/fisiología , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Selective transport of pyruvate across the inner mitochondrial membrane by the mitochondrial pyruvate carrier (MPC) is a fundamental step that couples cytosolic and mitochondrial metabolism. The recent molecular identification of the MPC complex has revealed two interacting subunits, MPC1 and MPC2. Although in yeast, an additional subunit, MPC3, can functionally replace MPC2, no alternative MPC subunits have been described in higher eukaryotes. Here, we report for the first time the existence of a novel MPC subunit termed MPC1-like (MPC1L), which is present uniquely in placental mammals. MPC1L shares high sequence, structural, and topological homology with MPC1. In addition, we provide several lines of evidence to show that MPC1L is functionally equivalent to MPC1: 1) when co-expressed with MPC2, it rescues pyruvate import in a MPC-deleted yeast strain; 2) in mammalian cells, it can associate with MPC2 to form a functional carrier as assessed by bioluminescence resonance energy transfer; 3) in MPC1 depleted mouse embryonic fibroblasts, MPC1L rescues the loss of pyruvate-driven respiration and stabilizes MPC2 expression; and 4) MPC1- and MPC1L-mediated pyruvate imports show similar efficiency. However, we show that MPC1L has a highly specific expression pattern and is localized almost exclusively in testis and more specifically in postmeiotic spermatids and sperm cells. This is in marked contrast to MPC1/MPC2, which are ubiquitously expressed throughout the organism. To date, the biological importance of this alternative MPC complex during spermatogenesis in placental mammals remains unknown. Nevertheless, these findings open up new avenues for investigating the structure-function relationship within the MPC complex.
Asunto(s)
Proteínas de Transporte de Anión/biosíntesis , Regulación de la Expresión Génica/fisiología , Proteínas de Transporte de Membrana Mitocondrial/biosíntesis , Espermátides/metabolismo , Espermatogénesis/fisiología , Testículo/metabolismo , Animales , Proteínas de Transporte de Anión/genética , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas Mitocondriales/biosíntesis , Proteínas Mitocondriales/genética , Transportadores de Ácidos Monocarboxílicos , Espermátides/citología , Testículo/citologíaRESUMEN
Mitochondria play a key role in energy metabolism, hosting the machinery for oxidative phosphorylation, the most efficient cellular pathway for generating ATP. A major checkpoint in this process is the transport of pyruvate produced by cytosolic glycolysis into the mitochondrial matrix, which is accomplished by the recently identified mitochondrial pyruvate carrier (MPC). As the gatekeeper for pyruvate entry into mitochondria, the MPC is thought to be of fundamental importance in establishing the metabolic programming of a cell. This is especially relevant in the context of the aerobic glycolysis, also known as the Warburg effect, which is a hallmark in many types of cancer, and MPC loss of function promotes cancer growth. Moreover, mitochondrial pyruvate uptake is needed for efficient hepatic gluconeogenesis and the regulation of blood glucose levels. In this review we discuss recent advances in our knowledge of the MPC, and we argue that it may offer a promising target in diseases like cancer and type 2 diabetes. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou.
Asunto(s)
Proteínas de Transporte de Anión/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Ácido Pirúvico/metabolismo , Animales , Proteínas de Transporte de Anión/deficiencia , Proteínas de Transporte de Anión/genética , Diabetes Mellitus Tipo 2/metabolismo , Proteínas de Drosophila/metabolismo , Metabolismo Energético , Glucosa/metabolismo , Homeostasis , Humanos , Hígado/metabolismo , Mamíferos/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/deficiencia , Proteínas de Transporte de Membrana Mitocondrial/genética , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales , Transportadores de Ácidos Monocarboxílicos , Neoplasias/metabolismo , Fosforilación Oxidativa , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Mitochondria contribute significantly to the cellular production of ROS. The deleterious effects of increased ROS levels have been implicated in a wide variety of pathological reactions. Apart from a direct detoxification of ROS molecules, protein quality control mechanisms are thought to protect protein functions in the presence of elevated ROS levels. The reactivities of molecular chaperones and proteases remove damaged polypeptides, maintaining enzyme activities, thereby contributing to cellular survival both under normal and stress conditions. We characterized the impact of oxidative stress on mitochondrial protein homeostasis by performing a proteomic analysis of isolated yeast mitochondria, determining the changes in protein abundance after ROS treatments. We identified a set of mitochondrial proteins as substrates of ROS-dependent proteolysis. Enzymes containing oxidation-sensitive prosthetic groups like iron/sulfur clusters represented major targets of stress-dependent degradation. We found that several proteins involved in ROS detoxification were also affected. We identified the ATP-dependent protease Pim1/LON as a major factor in the degradation of ROS-modified soluble polypeptides localized in the matrix compartment. As Pim1/LON expression was induced significantly under ROS treatment, we propose that this protease system performs a crucial protective function under oxidative stress conditions.
Asunto(s)
Homeostasis/fisiología , Proteínas Mitocondriales/metabolismo , Estrés Oxidativo/fisiología , Proteoma/metabolismo , Aconitato Hidratasa/metabolismo , Antimicina A/farmacología , Citocromo-c Peroxidasa/metabolismo , Hidroliasas/metabolismo , Peróxido de Hidrógeno/farmacología , Peroxirredoxinas/metabolismo , Proteoma/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Vitamina K 3/farmacologíaRESUMEN
Pyruvate metabolism plays a pivotal role in cell homeostasis and energy production. Pyruvate, the end product of glycolysis, is either catabolized in the cytosol, or enters into mitochondria to promote oxidative phosphorylation. The import of pyruvate into mitochondria requires a specific carrier in the inner mitochondrial membrane, the mitochondrial pyruvate carrier (MPC), whose identity was only recently discovered. Here we report our current knowledge of the structure and function of the MPC and we describe how dysfunction of the MPC could participate in various pathologies, including type 2 diabetes and cancer.
Asunto(s)
Homeostasis , Mitocondrias/metabolismo , Ácido Pirúvico/metabolismo , Animales , Glucosa/metabolismo , Humanos , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana Mitocondrial , Membranas Mitocondriales/metabolismo , Transportadores de Ácidos Monocarboxílicos , Mutación/genética , Neoplasias/metabolismo , Neoplasias/patologíaRESUMEN
Although mitochondria are usually considered as supporters of life, they are also involved in cellular death. Mitochondrial outer membrane permeabilization (MOMP) is a crucial event during apoptosis because it causes the release of proapoptotic factors from the mitochondrial intermembrane space to the cytosol. MOMP is mainly controlled by the Bcl-2 family of proteins, which consists of both proapoptotic and antiapoptotic members. We discuss the current understanding of how activating and inhibitory interactions within this family lead to the activation and oligomerization of MOMP effectors Bax and Bak, which result in membrane permeabilization. The order of events leading to MOMP is then highlighted step by step, emphasizing recent discoveries regarding the formation of Bax/Bak pores on the outer mitochondrial membrane. Besides the Bcl-2 proteins, the mitochondrial organelle contributes to and possibly regulates MOMP, because mitochondrial resident proteins and membrane lipids are prominently involved in the process.
Asunto(s)
Apoptosis/fisiología , Membranas Mitocondriales/fisiología , Modelos Biológicos , Lípidos de la Membrana/metabolismo , Lípidos de la Membrana/fisiología , Membranas Mitocondriales/ultraestructura , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/fisiología , Permeabilidad , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Transducción de SeñalRESUMEN
Proteins in a natural environment are constantly challenged by stress conditions, causing their destabilization, unfolding, and, ultimately, aggregation. Protein aggregation has been associated with a wide variety of pathological conditions, especially neurodegenerative disorders, stressing the importance of adequate cellular protein quality control measures to counteract aggregate formation. To secure protein homeostasis, mitochondria contain an elaborate protein quality control system, consisting of chaperones and ATP-dependent proteases. To determine the effects of protein aggregation on the functional integrity of mitochondria, we set out to identify aggregation-prone endogenous mitochondrial proteins. We could show that major metabolic pathways in mitochondria were affected by the aggregation of key enzyme components, which were largely inactivated after heat stress. Furthermore, treatment with elevated levels of reactive oxygen species strongly influenced the aggregation behavior, in particular in combination with elevated temperatures. Using specific chaperone mutant strains, we showed a protective effect of the mitochondrial Hsp70 and Hsp60 chaperone systems. Moreover, accumulation of aggregated polypeptides was strongly decreased by the AAA-protease Pim1/LON. We therefore propose that the proteolytic breakdown of aggregation-prone polypeptides represents a major protective strategy to prevent the in vivo formation of aggregates in mitochondria.
Asunto(s)
Proteasas ATP-Dependientes/metabolismo , Mitocondrias/enzimología , Proteínas Mitocondriales/metabolismo , Chaperonas Moleculares/metabolismo , Estructura Cuaternaria de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina Endopeptidasas/metabolismo , Estrés Fisiológico , Chaperonina 60/metabolismo , Activación Enzimática , Proteínas HSP70 de Choque Térmico/metabolismo , Respuesta al Choque Térmico , Cinética , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Saccharomyces cerevisiae/enzimologíaRESUMEN
The cellular role of Hsp100/Clp chaperones in maintaining protein stability is based on two functional aspects. Under normal growth conditions they represent components of cellular protein quality control machineries that selectively remove damaged or misfolded polypeptides in cooperation with specific proteases. After thermal stress, proteins of the ClpB subfamily have the unique ability to directly resolubilize aggregated polypeptides in concert with Hsp70-type chaperones, leading to the recovery of enzymatic activity. Hsp78, the homolog of the bacterial chaperone ClpB in mitochondria of eukaryotic organisms, participates in both protective activities. Hsp78 is involved in conferring thermotolerance to the mitochondrial compartment but also participates in protein degradation by the matrix protease Pim1. Despite the high sequence conservation between Hsp78 and ClpB, an analysis of the structural properties revealed significant differences. The identified mitochondrial Hsp78s do not contain N-terminal substrate-binding domains. In addition, formation of the oligomeric chaperone complex was more variable as anticipated from the studies with bacterial ClpB. Hsp78 predominantly formed a trimeric complex under in vivo conditions. Hence, mitochondrial Hsp78s form a distinct subgroup of the ClpB chaperone family, exhibiting specific structural and functional properties.