Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae

! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae Tânia Catarina da Silva Maia Medeiros Mestrado em Biologia Celular e Molecular Departamento de Biologia 2013 Orientador Doutor Vitor Manuel Vieira da Costa Instituto Ciências Biomédicas Abel Salazar II! FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae III! ! Todas as correções determinadas pelo júri, e só essas, foram efetuadas. O Presidente do Júri, Porto, ______/______/_________ IV! FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae! ! FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae V! ! Dissertação de candidatura ao grau de Mestre em Biologia Celular e Molecular submetida à Faculdade de Ciências da Universidade do Porto. O presente trabalho foi desenvolvido sob a orientação científica do Doutor Vitor Manuel Vieira da Costa e foi realizado no Instituto de Biologia Molecular e Celular da Universidade do Porto. Dissertation for applying to the Master’s Degree in Molecular and Cell Biology, submitted to the Faculty of Sciences of the University of Porto. The present work was developed under the scientific supervision of Doctor Vitor Manuel Vieira da Costa and was done at the Institute for Molecular and Cell Biology of the University of Porto. VI! FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae! ! FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae VII! ! Acknowledgments Costuma ser da praxe, agradecer a todas as pessoas, que tornaram a realização deste trabalho, há contributos de natureza diversa que não podem e nem devem deixar de ser realçados. E eu não fui exceção. Ao Professor Vitor Costa, meu orientador, inicialmente por me ter aceite no seu laboratório, pela competência científica e acompanhamento do trabalho, pela disponibilidade e generosidade revelada, assim como pelas críticas, correções e sugestões relevantes feitas durante a orientação. Ao Vitor Teixeira, por todo que me ENSINOU, por todas as CONVERSAS, por todos os PUXÕES de orelhas, pela sua DEDICAÇÂO, pela revisão ao longo de todo o trabalho. Por me fazer sentir capaz, e que sempre me estimulou a crescer científica e pessoalmente. A todas os elementos do RCS/MCA e MicroBiosyn, em especial: Rita Vilaça, companheira de bancada dos últimos tempos; Sílvia e Rute, as mãezinhas de serviço; às Catarinas e ao Paulo. Ao Ivan, não saberia o que dizer se tivesse que dizer grande coisa. Ao meu JORGINHO, e RITA, as pestinhas da casa que fazem com que a vida seja sempre cheia de sorrisos. Aos meus PAIS, e ao meu Padrinho FRANCISCO que me deram mais que apoio EMOCIONAL e financeiro. Comprometeram-se por um futuro melhor e simplesmente porque acreditaram. A ti Maria da Graça, que me ensinaste a viver, a perdoar, que me ensinaste a ser feliz. A ti minha madrinha, pelo secreto ADEUS! VIII! FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae! ! Resumo As mitocôndrias desempenham um importante papel na obtenção de energia através da fosforilação oxidativa. No entanto, são também responsáveis pela regulação de diversos processos biológicos nomeadamente a resposta ao stress e morte celular. Os esfingolipídos, tal como a ceramida, a esfingosina e a esfingosina-1-fosfato são componentes estruturais das membranas celulares e os seus metabolitos desempenham importantes funções reguladoras, que incluem a modelação de vias de sinalização e de uma série de processos celulares, incluindo a apoptose, proliferação, ciclo celular, respostas ao stress e o envelhecimento. Nos últimos anos, temos assistido a um grande interesse por parte da comunidade científica no estudo do papel dos esfingolípidos na função mitocondrial, homeostasia redox e envelhecimento, uma vez que o metabolismo dos esfingolípidos poderá ser terapeuticamente relevante no tratamento de patologias associadas com o envelhecimento. Na levedura Saccharomyces cerevisiae, a degradação de esfingolípidos complexos é catalizada pela enzima Isc1p, ortóloga da esfingomielinase neutra tipo 2 em mamíferos. Células com deficiência na proteína Isc1p apresentam alterações no metabolismo e função mitocondrial, hipersensibilidade ao peróxido de hidrogénio e envelhecimento cronológico prematuro. As disfunções mitocondriais das celulas isc1Δ estão associadas com uma sobrecarga de ferro e aumento de morte celular por apoptose induzida pelo peróxido de hidrogénio e envelhecimento cronológico. Estudos anteriores implicam a proteína cinase da família das proteínas AGC Sch9p nos fenótipos das células isc1Δ. Apesar da ativação da via ocorrer em resposta a nutrientes e sinais de stress por parte do complexo TORC1, a proteína Sch9p regula também a função mitocondrial e o envelhecimento cronológico integrando sinais de esfingolípidos. Como esta complexa via regula o envelhecimento cronológico, a resistência ao stress oxidativo, a função mitocondrial e a autofagia permanence muito pouco caraterizada. O trabalho apresentado nesta dissertação aborda o papel da esfingomielinase neutral Isc1p e a proteína cinase Sch9p na regulação da função mitochondrial e autofagia, e a sua revelância no stress oxidativo e envelhecimento cronológico. Os resultados demonstram que a deleção do gene SCH9 suprime a hipersensibilidade ao peróxido de hidrogénio e as disfunções mitocondriais das células isc1∆, tal como a incapacidade de crescer em fontes de carbono não fermentáveis como o glicerol, a baixa atividade da enzima citocromo c oxidase (COX) e o reduzido consumo de oxigénio. Por fim, também foi possível demonstrar que o mutante isc1Δ exibe um decréscimo no fluxo autofágico e uma hiperativação da FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae IX! ! mitofagia, em parte mediado pela proteína cinase Sch9p. Os resultados obtidos sugerem que a ativação da proteina cinase Sch9p desempenha um importante papel nas disfunções mitocondriais e na desregulação da autofagia e mitofagia em células isc1Δ, contribuindo desta forma para a hipersensibilidade ao stress oxidativo e ao envlhecimento cronológico prematuro exibido por este mutante. Palavras chave: esfingolípidos, função mitocondrial, stress oxidativo, longevidade, Isc1p, Sch9p. XVI! FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae! ! M(IP)2C Mannose diinositolphosphoryl-ceramide NADPH Nicotinamide adenine dinucleotide phosphate nSMase Neutral sphingomyelinase OD Optical density PAGE Polyacrylamide gel electrophorese PBS Phosphate buffered saline PCR Polymerase chain reaction PHS Phytosphingosine PHS-1-P Phytosphingosine-1-phosphate ROS Reactive oxygen species SD Standard deviation SDS Sodium dodecyl sulfate SM Sphingomyelin SMase Sphingomyelinase SPT Serine palmitoyltransferase S1P Sphingosine-1-phosphate S6K Ribosomal protein S6 kinase TBS Tris buffered saline TOR Target of Rapamycin TORC1 Target of Rapamycin Complex 1 TTBS Tris-buffered saline plus Tween UV Ultraviolet YPD Yeast peptone dextrose FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae XVII! ! YPG Yeast peptone glycerol XVIII! FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae! ! FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae 1 ! Chapter I Introduction ! 2! FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae! ! I-1. Saccharomyces cerevisiae as a biological model to study basic cellular processes The budding yeast Saccharomyces cerevisiae is a unicellular fungus of the Ascomycete family with approximately 6000 genes. Due to the high conservation of fundamental biochemical pathways, yeast has been used as a model organism to unravel new aspects of important biological processes in higher eukaryotes (Mager and Winderickx, 2005). The studies of aging (Longo, 2003; Piper, 2006; Barros et al., 2010), cell cycle (Humphrey and Pearce, 2005), stress responses (Costa et al., 2007; Rodrigues-Pousada et al., 2010) or apoptosis (Almeida et al., 2008; Carmona-Gutierrez et al., 2010; Greenwood and Ludovico, 2010) have served in many ways to foster our understanding about these processes. Among all eukaryotic model organisms, S. cerevisiae combines several advantages. For instance, it can be cultured in different media, it has a short doubling time, and it has a convenient experimental tractability, due to simple growth conditions and easy genetic manipulations (Mager and Winderickx, 2005). The yeast model has well established genomic and proteomic methodologies and there are well-curated databases that provide overall information about protein-protein interactions, genetic interactions, protein function and predicted orthologues in higher organisms (Mager and Winderickx, 2005; Pena-Castillo and Hughes, 2007; Petranovic and Nielsen, 2008). The elucidation of sphingolipid metabolism and dynamics in yeast cells and their route of synthesis have been important to uncover new functions of sphingolipids and to understand the mechanisms for sphingolipid homeostasis in both physiological and pathological conditions. The budding yeast has been used to identify nearly all of the genes that encode sphingolipid metabolic enzymes and many of these were critical in identifying mammalian homologs (Dickson and Lester, 2002; Sims et al., 2004.), showing that yeast and mammals share many similarities in sphingolipid metabolism. Therefore, the budding yeast is considered to be a useful model organism to study sphingolipid metabolism and regulation. FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae 3 ! I-2. An overview of sphingolipid structure and bioactivity !I-2.1. Structure Sphingolipids are important structural components of cell membranes found in essentially all animals, plants and fungi, as well as some prokaryotic organisms (Merrill et al., 2007). They are mostly found on the outer leaflet of the plasma membrane, although they are also present at membranes of different organelles at variable ratio. In addition, they are major constituents of lipoproteins. Several species of sphingolipids have been identified and some of them are bioactive lipids since they have the ability to modulate signalling pathways and the variation of their ratio results in important modifications in cellular functions and fate (Hannun and Obeid, 2008). In fact, sphingolipids such as sphingosine, ceramide and sphingosine-1-phosphate have emerged as core sphingolipids in this metabolism as they regulate a vast number of cellular processes, including cell growth, adhesion, migration, senescence, apoptosis, and autophagy (Hannun and Obeid, 2008; Ryland et al., 2011). From a structural point of view, sphingolipids have an amphipathic nature and are composed by a long chain sphingoid base (LCB), generally 18 carbons long (sphingosine), with the C2amino group amide-linked to a fatty acid, thereby forming the core unit, to which polar groups are added to form different types of sphingolipids (Malagarie et al., 2002, figure 1). The nature of the fatty acid (carbon length, degree of unsaturation and hydroxylation) along with other modifications of the long-chain bases and the polar head group define the vast family of sphingolipids (Merrill et al., 2007; Hannun and Obeid, 2008). ! ! Figure -1. General sphingolipid structure. The image was modified from refrence (Malagarie et al., 2002). ! 4! FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae! ! I-2.2 Bioactive sphingolipids and regulation of biological processes The complexity of sphingolipid metabolism arises from the interconnectivity of bioactive lipids (Hannun and Obeid, 2008), which enable cells to orchestrate different cellular responses by regulating sphingolipid interconversions (figure 2). ! Figure-2. Overview of sphingolpid metabolism and interconnectivity of bioactive sphingolipids. The image was obtained from reference (Hannun and Obeid, 2008). In response to both extracellular stress (e.g., UV, hypoxia, toxins, heat stress) and alterations in cell physiology, the enzymes involved in sphingolipid metabolism act in a coordinate manner to regulate not only the levels of individual bioactive lipids, but also their metabolic interconversion (figure 3). FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae 5 ! Figure-3. An overview of the roles of sphingolipids in Biology. The image was obtained from reference (Hannun and Obeid, 2008). The sphingolipids ceramide, sphingosine and sphingosine-1-phosphate (S1P) are the main representatives of sphingolipid metabolism and play crucial roles in the regulation of many cellular processes (Hannun and Obeid, 2008). The first sphingolipid to be identified was sphingosine and it exerts pleiotropic effects on protein kinases and other targets (Hannun et al., 1986). Sphingosine and its related sphingoid bases have roles in regulating the actin cytoskeleton, endocytosis, cell cycle and apoptosis (Smith et al., 2000). Ceramide mediates many cell-stress responses that include the regulation of apoptosis (Obeid et al., 1993) and cell senescence (Venable et al., 1995), by modulating the activity of ceramide-activated protein kinases (e.g. PKC) and phosphatases (CAPP, PP1 and PP2A) (figure 3). On the other hand, S1P promotes cell proliferation and survival by acting in an autocrine manner on S1P receptors (Hla, 2004). Consequently, it is expected that alterations in the relative 6! FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae! ! amounts of sphingosine-1-phosphate and sphingosine/ceramide have significant effects on cell physiology and metabolism and ultimately on cell fate (Spiegel and Milstein, 2003). Other components of the family of sphingolipids include ceramide-1!phosphate (C1P), which is involved in inflammation and vesicular trafficking, glucosylceramide, mostly associated with post-Golgi trafficking and drug resistance, lyso-sphingomyelin and dihydroceramide (Hannun and Obeid, 2008). The importance of sphingolipid signalling derives form the early recognition of their contribution in the pathobiology of human cancers and other human ailments such as diabetes and heart disease, microbial infections, neurological and immune dysfunctions (Kolter and Sandhoff, 2006; Ozbayraktar and Ulgen, 2009; Kolter, 2011; Hla and Dannenberg, 2012; Young et al., 2013). FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae 7 ! I-3. Yeast sphingolipid metabolism The general pathways governing sphingolipid metabolism are well characterized in yeast (figure 4). It is very similar to the mammalian counterpart (figure 2), and shares a similar spatial organization, with the early steps taking place in the endoplasmic reticulum (ER) and the subsequent processes occurring in the Golgi compartment for the synthesis of more complex sphingolipids (Futerman and Riezman, 2005). ! Figure-4. Schematic overview of yeast sphingolipid metabolism displaying the metabolic intermediates, genes involved and cell location of the enzimatic reactions. The image was modified from reference (Vallee and Riezman, 2005). 14! FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae! ! Figure-5. Crosstalk between nutrient and sphingolipids signalling pathways that control mitochondrial function, redox homeostasis and lifespan in yeast. TORC1 is activated by nutrients. This protein activates Sch9p by phosphorylation at the C-terminus. In addition, Sch9p is phosphorylated in a Thr570 residue in the activation loop by Pkh1/2p protein kinases in response to LCBs. Sch9p governs redox homeostasis and lifespan by acting as a physiological core center integrating nutrient and stress signal from TORC1 and sphingolipid signalling derived from LCB-Pkh1/2p axis. Ceramide-mediated activation of the Sit4p protein phosphatase may also play roles in regulating lifespan. Adapted from reference (Huang et al., 2012). Additionally, TORC1 plays a major role in regulation of autophagy (Yorimitsu et al, 2007), a major lysosomal/vacuolar degradative pathway for bulk proteins and damaged and/or unnecessary organelles (Mizushima and Klionsky, 2007). How this signaling pathway coordinate with sphingolipid dynamics in the regulation of cell metabolism and survival remains poorly characterized. I-5. Autophagy In order to maintain viability during starvation periods, yeast undergoes a degradative process of its own cellular components by a "self-eating" process via the vacuole named autophagy (Klionski and Erm, 2000). Autophagy is an evolutionarily conserved process in eukaryotic cells that involves the engulfment of cytoplasmic cargo into double-membrane organelles called autophagosomes. After their formation, autophagosomes fuse with lysosomes (or the vacuole in yeast), within which the inner membrane and the cargo are degraded (Mizushima, 2007; Klionsky et al., 2007; Nakatogawa et al., 2009). A basal level of constitutive autophagy is crucial for routine clearance of the cytosol under normal conditions. Basal autophagy is critical for protein and organelle homeostasis and FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae 15 ! quality control in post-mitotic differentiated cells, such as neurons (Mizushima and Levin, 2010). In addition, autophagy becomes activated in response to low nutrient availability, (nitrogen and carbon starvation) providing a source of nutrients and energy (Blommaart et al., 1997; Mizushima et al., 2002). Autophagy is also triggered as an adaptive response to a broad range of other extracellular or intracellular stressors such as hypoxia, heat, reactive oxygen species (ROS) and accumulation of damaged cytoplasmic components (Levine and Klionsky, 2004). Three major subtypes of the autophagy have been described: macroautophagy (the most common subtype), microautophagy, and chaperone-mediated autophagy (Ravikumar et al., 2010). In addition, a number of specific subtypes exist. The different forms of autophagy are shown in figure 6 and are discussed below in more detail. ! Figure-6. Different autophagic-like processes in cell metabolism and physiology. Macroautophagy, microautophagy (selective degradation of organelles) and chaperone-mediated autophagy (CMA) are shown. The image was altered from (Yen and Klionsky, 2008). (i) Macroautophagy, where proteins or entire organelles are engulfed in a double membrane vesicle termed the autophagosome and subsequently degraded by vacuole enzymes, is the most prevalent form of autophagy and will be herein referred as to autophagy. Macroautophagy plays many roles in the cell, namely in starvation adaptation and metabolism as well as development and differentiation (Yang and Klionski, 2009; Farre et al., 2009; Kroemer, et al., 2010; Ravikumar et al., 2010;); 16! FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae! ! (ii) Microautophagy is a process in which cytoplasm is directly engulfed at the surface of the degradative organelle (the vacuole or lysosome) without the production of autophagosomes. The membrane invaginates, and pinches off to form an internal autophagic vesicle containing cytoplasmic material (Kunz et al., 2004). The selective autophagy of particular organelles has been described, for example “mitophagy” is the selective degradation of mitochondria by autophagy, and “pexophagy” describes the selective turnover of peroxisomes by microor macroautophagy and ribophagy the selective turnover of ribosomes (figure 6) (Tuttle and Dunn, 1995; Dunn et al., 2005; Kanki and Klionsky, 2008). (iii) Chaperone-mediated autophagy (CMA) is a selective form of autophagy, so far only detected in mammalian cells, that is activated during long-term nutrient deprivation. CMA does not involve the formation of a double membrane vesicle and targets chaperones to proteins that contain a motif biochemically related to the pentapeptide KFERQ. The chaperone-KFERQ-containing protein complex then binds LAMP (lysosome-associated membrane protein)- 2A receptors on the lysosome membrane, and translocates the target protein into the lysosomes for degradation (review in Bejarana and Cuervo, 2010). Finally, the cytoplasm to vacuole (Cvt) targeting pathway is an example of a selective, autophagy-like pathway that is specific to yeast, in which the hydrolases aminopeptidase 1 and α-mannosidase are selectively transported to the vacuole (Huang and Klionsky, 2002). Thirty-five autophagy-related genes (ATG) in yeast have been so far identified, and, many of them present homologues in higher eukaryotes (Yang and Klionsky, 2009). ATG proteins are organized in functional complexes that mediate the diverse steps of macroautophagy and other selective forms of autophagy: induction/initiation, vesicle nucleation, cargo recognition and packaging, vesicle expansion and sealing, fusion with the lysosome, vesicle breakdown and recycling of the resulting macromolecules (figure 7). FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae 17 ! ! Figure-7. Schematic representation of autophagy. Autophagy undergoes several processes: nucleation, elongation, formation of autophagosomes, maturation, formation of autolysosomes and degradation of cargo. The image was obtained from (Kraft and Martens, 2012). I-5.1. Autophagy and signalling pathways Some signalling pathways have been characterized as playing a role in the regulation of autophagy. The regulatory proteins of these pathways are the Target of Rapamycin (TOR), Sch9p, Ras/cAMP-dependent protein kinase A (PKA), and Pho85p (Budovskaya et al., 2004; Yorimitsu et al., 2007; Yang et al., 2010). Under nutrient-rich conditions, autophagy is inhibited because TORC1 is activated and drives the hyperphosphorylation of the protein Atg13p, resulting in a lower affinity for Atg1p and Atg17p to begin the induction of the process (figure 8) (Kamada et al., 2010). In this process, it is also known that PKA and Sch9p are involved in the regulation of Atg13p phosphorylation and localization to the preautophagosomal structure (Stephan et al., 2009), although the mechanisms involved are yet to be understood. When TORC1 activity is inhibited, either by rapamycin or starvation, Atg13p is rapidly dephosphorylated (to yield a hypo-phosphorylated form of Atg13p) and can interact with the Atg1p serine/threonine kinase. The Atg1-Atg13 protein complex then associates with Atg17p, which is part of a ternary complex with Atg29p and Atg31p (Cebollero and Reggiori, 2009; Nakatogawa et al., 2009; Chang and Neufeld, 2010; Kamada et al., 2010). The Atg1Atg13 protein complex then recruits other Atg proteins to the phagophore assembly site (PAS) and controls their dynamics (Kabeya et al., 2005; Cheong et al., 2008, Kawamata et 18! FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae! ! al., 2008). Autophagosome nucleation requires a complex containing Atg6p and the class III phosphatidylinositol 3-kinase Vps34p, the latter generating phosphatidylinositol 3phosphate. Figure-8. Schematic diagram of the various stages of autophagy. Stage 1 involves the regulation of autophagy induction, in which mTOR is inactivated, allowing for the activation of the Ulk1 kinase complex. In stage 2, nucleation, the Class III PI(3)K complex forms which is necessary for formation of the isolation membrane. The membrane expands to engulf cytosolic contents. In stage 3, vesicle elongation, a process that requires the two ubiquitin-like conjugation steps of Atg5–Atg12 and LC3/Atg8p–PE. In stage 4, vesicle retrieval, the transport of Atg9p between the PAS and non-PAS sites is necessary for autophagosome formation and requires Atg18. Stage 5, vesicle maturation, involves trafficking and fusion of the fully enclosed double-membrane autophagosome to various endosomal compartments, which finally fuses with the lysosome to form the autolysosome. In the final stage, stage 6, degradation (the contents of the autolysosome are degraded by resident lysosomal enzymes). Although the process is described for mammalian cells, similar features are also observed for yeast cells. The image was obtained from (Maiuri et al., 2007). The expansion of autophagosomal membranes involves two ubiquitin-like molecules, Atg12p and Atg8p, an E1 ubiquitin activating enzyme (Atg7p), two analogues of ubiquitin-conjugated enzymes (Atg10p and Atg3p), an Atg8p modifying protease (Atg4p), the protein target of Atg12p attachment (Atg5p) and Atg16p. In the first ubiquitination reaction, the E1-like Atg7p and the E2-like Atg10p promote the association of Atg12p with Atg5p (Suzuki et al., 2001; Suziki et al., 2007). This conjugate subsequently interacts with Atg16p to generate preautophagosomal structures (PAS) (Mizushima et al., 1999). In the second ubiquitin reaction, Atg8p is cleaved by the protease Atg4p and conjugated to phosphatidylethanolamine (PE) by Atg7p (E1-like) and Atg3p (E2-like) (Kim et al., 1999; FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae 19 ! Kirisako et al., 2000;). This lipidated form of Atg8p is essential to drive proper autophagosome biogenesis (Nair et al., 2012). Upon completion of autophagosome formation, the Atg12–Atg5–Atg16 protein complex is released into the cytosol, whereas Atg8-PE remains stably associated with the autophagosomal membranes (Kirisaki et al., 2000). Lysosome docking and fusion occurs when the outer autophagosomal membrane fuses with the lysosomal membrane to produce an autophagic body (autolysosome in mammalian cells). The remaining single-membrane that envelops the cargo is lysed and the population of Atg8-PE together with the enclosed cargo are released into the lysosome lumen and degraded by resident vacuolar hydrolases (proteases, lipases, nucleases and glucosidases) (Kirisako et al., 1999; Kabeba et al., 2000). The resulting degradation products are released back into the cytosol through the activity of specific membrane permeases for recycling. I-5.2. Autophagy and aging The relationship between CLS and autophagy is extremely complex and not fully understood. Autophagy appears to be a common downstream target of multiple cellular pathways with well-known roles in longevity regulation (Madeo et al., 2010). The upregulation of autophagy extends chronological lifespan in mice, Caenorhabditis elegans, yeast and other organisms (Eisenberg et al., 2009). Importantly, the TOR/Sch9p and the Ras/cAMP-dependent protein kinase proteins, which integrate the network of nutrientsensing pathways and regulate autophagy, are known to be involved in proper regulation of longevity pathways (Kaeberlein et al., 2005; Gomes et al., 2007; Hen and Klionsky, 2011). Recently, Hansen et al. found that dietary restriction and TOR inhibition in C. elegans produce an autophagic phenotype and that inhibiting genes required for autophagy prevents dietary restriction and TOR inhibition from extending lifespan, corroborating with this conception (Hansen et al., 2008). Screenings performed in yeast have demonstrated that genes encoding proteins of the autophagic machinery are necessary to extend lifespan during nitrogen starvation (Tsukada, 1993). Suppression of autophagy by knockdown of essential autophagy genes triggers apoptosis or necrosis in cells that would otherwise survive under stress conditions (reviewed in Kourtis and Tavernarakis, 2009; Mathew et al., 2009). Autophagy appears to serve primarily a cytoprotective function by maintaining nutrient and energy homeostasis during starvation or by degrading damaged cellular components and invasive pathogens (review in Lionaki et al., 2013). Paradoxically, although autophagy is a predominantly homeostatic mechanism, it can also play a role in cell death, which is not restricted to developmental 20! FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae! ! programmed cell death, but extends to cell death that occurs in many pathological conditions. Excessive autophagy induced by extreme conditions such as toxins and necrosis-triggering insults might cause uncontrollable degradation or sequestration of cells contents resulting in undesirable cell death if not properly regulated (Samara and Tavernarakis, 2008; Kourtis and Tavernarakis, 2009; Yang and Klionsky, 2010). I-5.3. Mitophagy: the autophagic-like selective degradation of mitochondria process! The view of mitochondrial dynamics has expanded into an integral cell biological process influencing many cellular functions and ultimately contributing to cell death and aging (Braun and Westermann, 2011). Mitochondria are dynamic structures that migrate throughout the cell, fuse and divide, and undergo regulated turnover (Westermann, 2010). On this basis, the regulation of mitochondrial dynamics (fusion/fission cycles) and the selective degradation of mitochondria by an-autophagic-like process (mitophagy) are important on the regulation of mitochondrial function and cell physiology by allowing mitochondrial recruitment to critical subcellular compartments, mitochondrial communication, regulation of the mitochondrial shape and to the mitochondrial quality control (Liesa and Shirihai, 2013). The mitochondrial theory of aging predicts that an accumulation of oxidative stress and mtDNA mutations eventually is associated with the onset of age-associated pathologies and cell death (Cadenas and Davies, 2000). Apparently, mitophagy is associated with the removal of damaged/dysfunctional or oxidized mitochondria and therefore contributes to the homeostatic maintenance of sustainable mitochondrial function, allowing an efficient process for ATP production and cellular energetics (Kissova et al., 2004). There are several lines of evidence in yeast studies suggesting that damaged mitochondria are eliminated by mitophagy. For example, interference with F1Fo-ATPase biogenesis in a temperature sensitive fmc1 mutant (Priault et al., 2005), or osmotic swelling of mitochondria caused by depletion of the mitochondrial K+/H+ exchanger Mdm38 (Nowikovsky et al., 2007) induce mitophagy. It is also conceivable to assume that this process allows complementation of mtDNA gene products in heteroplasmic cells that have accumulated different somatic mutations, thus diluting the effect of mtDNA mutations and depolarized mitochondria during aging. Furthermore, Mao et al. have recently disclosed an important link between mitophagy and mitochondrial dynamics. On this basis, both processes may act in a coordinate manner to assure the proper connectivity of the mitochondrial network, which is an important factor that determines the cell’s response to calcium and other pro-apoptotic signals and ultimately cell fate (Mao et al., 2011). In addition, mitophagy is also an essential step in certain FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae 21 ! developmental processes such as embryonic development and spermatogenesis (Al Rawi et al., 2011; Sato and Sato, 2011). The core autophagic machinery used is common with other types of autophagy. The requirement of several ATG genes for mitophagy has been reported from several groups (Kissova et al., 2004; 2007; Tal et al., 2007; Zhang et al., 2007; Kanki and Klionsky, 2008) and some atg mutants strains in S. cerevisiae screenings were identified to be selectively involved in mitophagy, namely ATG32 and ATG33 genes (Kanki et al., 2009). Their function is not completely understood in the process. In yeast studies, there are several ways to induced mitophagy. The most common are the incubation in nitrogen starvation conditions (Kissova et al., 2007; Mao et al., 2011; Suzuki et al., 2011, Kurihara et al., 2012), treatment with the TORC1 inhibitor, rapamycin, after preculturing yeast in a non-fermentable medium that induces the proliferation of mitochondria (e.g. lactate) (Tal et al., 2007; Kanki and Klionsky, 2008; Kanki et al., 2009). In more physiological conditions, mitophagy is induced at stationary phase when yeast cells are cultured in a medium with a non-fermentable carbon source (Tal et al., 2007; Kanki and Klionsky, 2008). Mitophagy has recently become the subject of much scientific interest. This is due in part to the central role of this organelle in various cellular processes, as well as the association of mitochondrial dysfunction with pathological conditions in humans such as the neurodegenerative Alzheimer’s and Parkinson’s diseases (Abeliovich, 2010). 22! FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae! ! FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae 23 ! Chapter II Aim of the work 30! FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae! ! during 30 cycles. PCR products were analyzed in 1% (wt/vol) agarose gel using 0,55µg/mL EtBr and TAE 1x as buffer, and DNA bands were compared to Gene Ruler Ladder Mix (Thermo Scientific) III-4. Gene disruption The disruption of ISC1 using LEU2 cassette was performed by homologue recombination in sch9∆. The deletion fragment containing LEU2 and the flanking regions of ISC1 was amplified from genomic DNA isolated from the BY4741 isc1Δ::LEU2 strain stored in the lab using primers ISC1_Amp_Fw and ISC1_Amp_Rv (Table 2). The disruption of ISC1 using a URA3 cassette was amplified from genomic DNA isolated from the BY4741 isc1Δ::URA3 strain stored in the lab using the same primers as described above. The purification of DNA from TAE agarose gels was performed with GFXTM PCR DNA and Gel Band Purification Kit (GE Healthcare). Cells were transformed by electroporation and selected in minimal medium lacking leucine and uracil, respectively. Gene disruption was confirmed by PCR (figure 9), using the following pair of primers: LEU2_Conf_Fw + LEU2_Conf_Rv and URA3_Conf_Fw + URA3_Conf_Rv, respectively (Table 2). The disruption of the PHO8 gene in BY4741, isc1Δ, sch9Δ and isc1Δsch9Δ cells was performed using a deletion fragment that contains a hygromycin cassette and the flanking regions of PHO8, as reported (Sampaio-Marques et al., 2012). The deletion fragment was amplified by PCR using the next set of primers: Pho8_Amp_Fw + Pho8_HPH_Rv and Pho8_Amp_Rv + Pho8_HPH_Fw (Table 2). Cells were transformed by electroporation and selected in YPD medium supplemented with hygromycin (150 µg/mL). The correct insertion of cassette was confirmed by PCR using the subsequent set of primers: Pho8_Conf_Fw + Pho8_HPH_Rv and Pho8_Conf_Rv + Pho8_HPH_Fw. These strains were then transformed with plasmid pYX242-mtPHO8 and selected in minimal medium lacking leucine. For the analysis of mitochondrial morphology, yeast cells were transformed with a plasmid expressing mitochondrial DsRed (pYX222-mtDsRed) and selected in minimal medium lacking histidine. For autophagy analysis, BY4741, isc1∆, sch9∆ and isc1∆sch9∆ were transformed with pRS416-GFP-ATG8 and selected in minimal medium lacking uracil. FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae 31 ! Table 2. Primers used in this work *Fw-Forward primer/Rv-Reverse primer Figure-9. General scheme of the strategy for the construction of yeast mutants. Step 1 represents the procedure used for the generation of the LEU2 cassette for the disruption of the ISC1 gene, step 2 accounts for the homologous recombination mechanism for proper integration on the desired region and the step 3 exemplifies the comfirmation of the correct integration of the disruption cassette on the genome. Primers Sequence ISC1_Amp_Fw 5´-CTTTCCGCGTAAAAAGGGAA-3´ ISC1_Amp_Rv 5´-TTGCTTTGCATCTATTGACGA-3´ LEU2_Conf_Fw 5´-AGACGATTGCTAACCACCTA-3´ LEU2_Conf_Rv 5´-CGAACGAGGCAGTAGTCATGTT-3´ URA3_Conf_Fw 5´-ATCATCGCCGAATACGAAAC-3´ URA3_Conf_Rv 5´-CCCGCAGAGTACTGCAATTT-3´ Pho8_Amp_Fw 5´-GCCAGCAAGTGGCTACATAAA-3´ Pho8_HPH_Rv 5´-AAAGCATCAGCTCATCGAGA-3´ Pho8_Amp_Rv 5´-CAGTACGTGTCATGCGGTTAG-3´ Pho8_HPH_Fw 5´-CGCAAGGAATCGGTCAATAC-3´ Pho8_Conf_Fw 5´-CGACATGAATAGCAGCATTGA-3´ Pho8_Conf_Rv 5´-TCACGCTATAGAATGCACCT-3´ 32! FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae! ! III-5. Yeast electroporation III_5.1. Preparation of electro-competent cells Cells were grown in 50 mL of YPD medium to an OD600= 1.3 – 1.5, harvested, and ressuspended in 10 mL of a solution containing 10 mM Tris-HCl, 1 mM EDTA, 100 mM lithium acetate, pH 7.5, and gently shacked during 45 min at 26ºC. Then, 250 µL of 1 M DTT was added and cells were shacked for 15 min at 26ºC. Ice-cold sterile water was added for a final volume of 50 mL and cells were centrifuged at 4ºC. Cells were firstly washed with 25 mL of ice-cold sterile water and then ressuspended in 50 µL of 1 M sorbitol (maintained at 4ºC). III-5.2. Electro-transformation and plating Electro-competent cells (40 µL) were mixed with 5 µL of deletion fragment (containing approximately 0.5 µg of DNA). The mixture was transferred to pre-chilled sterile 2 mm electroporation cuvette. An electric pulse (1.5 kV, 25 µF and 200 Ω) was applied in parallel using an electroporation system (BioRad). After the pulse delivery, 1 mL of selective minimal medium was immediately added and cells were allowed to recover in appropriate media for 30 min (replicative plasmids) or 4 hours (integrative cassettes) at 26 ºC. Cells were then plated in selective medium and grown for 3 days at 26 ºC. III-6. Oxidative stress resistance For the analysis of oxidative stress resistance, yeast cells were grown in SC-medium to exponential phase (OD600=0.6) and treated with H2O2 (Merck) for 1 hour. Cell viability was determined by standard dilution plate counts on YPD medium containing 1.5 % agar (w/v). Colonies were counted after growth at 26 ºC for 3 days. Viability was expressed as the percentage of the colony-forming units (CFUs) (treated cells vs. untreated cells). III-7. Enzymatic activities and oxygen consumption For enzymatic activities, yeast cells were harvested by centrifugation for 5 min at 4000 rpm (4ºC). Cells were then ressuspended in 50 mM potassium phosphate buffer (pH 7.0) FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae 33 ! containing protease inhibitors (Complete, Mini, EDTA-free Protease Cocktail Inhibitor Tablets; Boehringer Mannhein) and total protein extracts were obtained by mechanical disruption through vigorous shaking of the cell suspension in the presence of glass beads for 5 min. Short pulses of 1 min were applied followed by 1-min incubation on ice. Cell debris was removed by centrifugation at 13000 rpm for 15 min and protein content was determined by the method of Lowry, using bovine serum albumin as a standard. Catalase activity was analyzed in situ, in the presence of 3,3′-diaminobenzidine tetrahydrochloride, using the H2O2/peroxidase system (Conyers and Kidwell, 1991). Superoxide dismutase activity was determined in situ, as described by e Flohe and Otting (1984). Cytochrome c oxidase (COX) activity was determined by measuring cytochrome c oxidation (Poyton et al., 1995). Oxygen consumption rate was measured for 3 x 108 cells in PBS buffer (pH 7.4), using an oxygen electrode (Oxygraph, Hansatech). Data was analyzed using the Oxyg32 V2.25 software. III-8. Mitochondrial membrane potential and ROS levels The mitochondrial membrane potential was assessed by flow cytometry, using cells labeled with 3,3’-dihexyloxacarbocyanine iodide (DiOC6(3), Molecular Probes), a mitochondrialspecific voltage-dependent dye (Rottenberg and Wu, 1998). Briefly, 2x106 cells were ressuspended in sample buffer [10 mM 2-(N-morpholino) ethanesulfonic acid, 0.1 mM MgCl2 and 2% (w/v) glucose, pH 6.0]. DiOC6(3) was added to a final concentration of 1 nM. The cell suspension was then incubated for 30 min at 26°C, collected by centrifugation and washed twice with PBS. Fluorescence was measured on the FL-1 channel with excitation and emission settings of 488 nm and 525 nm, respectively, without compensation. Data was analyzed using FlowJo software (Tree Star). For the quantification of ROS levels, 5x106 cells were ressuspended in PBS and the superoxide anion sensitive probe dihydroethidium (DHE, Molecular Probes) was added to a final concentration of 5 µM. Cells were incubated for 10 min at 26°C, pelleted by centrifugation, washed twice with PBS and analyzed by flow cytometry with excitation and emission settings of 488 nm and ≥670 nm (FL-3 channel), without compensation. Data was analyzed using FlowJo software (Tree Star). 34! FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae! ! III-9. Fluorescence microscopy For mitochondrial morphology analysis, cells carrying the plasmid expressing a mitochondria-targeted DsRed fluorescent protein (pYX222-mtDsRed) were grown in SCmedium lacking histidine to the post-diauxic shift phase. Live cells were observed by fluorescence microscopy (AxioImager Z1, Carl Zeiss). Data image stacks were deconvolved by QMLE algorithm of Huygens Professional v3.0.2p1 (Scientific Volume Imaging B.V.). Maximum intensity projection was used to output final images using ImageJ 1.45v software. III-10. Western blot analysis To assess alterations in autophagic flux, cells harboring the plasmid pRS416-GFP-ATG8 were grown to the exponential phase in SC-medium lacking uracil and treated with either rapamycin (200 ng/mL, (Sigma-Aldrich)) or DMSO (vehicle, Sigma-Aldrich) for 3 h. Total protein extracts (30 µg) were separated by SDS-PAGE using 10% SDS-polyacrylamide gels at 16 mA and transferred to a nitrocellulose membrane (Hybond-ECL, GE Healthcare) at 0.8 mA/cm2 during 1 h. After blotting, the nitrocellulose membranes were stained with Ponceau S (0,6 % (wt/vol) of Ponceau S, 3 % (wt/vol) TCA and 3 % (wt/vol) sulfosalicylic acid acid) to visualize proteins. Membranes were blocked for at least 2 h in TTBS [TBS supplemented with 0.05% (v/v) Tween-20 (Merck)] containing 5 % (w/v) non-fat dry milk and then incubated overnight with the primary antibody anti-GFP (1:3000; Roche). After washing twice with TTBS for 15 min, membranes were incubated with the secondary α-mouse IgG (1:3000; Molecular Probes) for 1 hour and then washed with TTBS and TBS twice. Immunodetection of bands was revealed by chemiluminescence, using a kit from GE Healthcare (RPN 2109). III-11. Alkaline phosphatase assay For the alkaline phosphatase assay, cells were harvested and ressuspended in 100 µL of assay buffer (250 mM Tris, 10 mM MgSO4, 10 mM ZnSO4, pH 9.0) and supplemented with 5 µL of Complete Mini protease inhibitor cocktail. The cells were lysed by vortexing with glass beads for 5 min. After centrifugation at 14000 rpm for 15 min, the supernatant was collected and the protein concentration was measured by the method of Lowry, using bovine serum albumin as a standard. 10 µg of total protein extract was added to reaction buffer (250 mM FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae 35 ! Tris, 10 mM MgSO4, 10 mM ZnSO4, 4.56 mM nitrophenyl-phosphate). Samples were incubated for 15 min at 30°C before terminating the reaction by adding 500 µL of stop buffer (2 M glycine, pH 11.0). The production of nitrophenol was monitored by measuring the absorbance at 400 nm, and the nitrophenol concentration was calculated using Beer’s law with ε400 = 18,000 M-1cm-1. One activity unit was defined as nmol nitrophenol/min/mg protein. III-12. Statistical analyses Data were analysed in GraphPad Prism Software v5.01 (GraphPad Software) and expressed as mean values ± SD from at least three independent experiments. Values were compared by Student’s t-test p< 0.05; **, p< 0.01; ***, p< 0.001; ****, p<0.0001 or two-way ANOVA with Bonferroni correction, as properly referred. 36! FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae! ! FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae 37 ! ! Chapter IV Results ! ! ! 38! FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae! ! IV-1. Characterization of oxidative stress resistance response IV-1.1. SCH9 disruption suppresses the oxidative stress sensitivity of isc1Δ cells ! Our lab has recently demonstrated that TORC1 activity is increased in Isc1p-deficient cells, as monitored by the TORC1-dependent phosphorylation of Sch9p at the C-terminus (Teixeira et al., unpublished results). Since Sch9p is a downstream effector of TORC1 in the regulation of mitochondrial function, oxidative stress and chronological lifespan in yeast (Wei et al., 2008) we evaluated if SCH9 deletion could abolish isc1Δ phenotypes, such as oxidative stress hypersensitivity and mitochondrial dysfunctions (Almeida et al., 2008). To assess oxidative stress resistance, cells were grown in SC-medium to the exponential (fermentative) phase and treated with 1.5 mM H2O2 for 1 h. As reported, sch9Δ cells were more resistant to oxidative stress than parental cells (Wei et al., 2008). Moreover, the deletion of SCH9 suppressed the hydrogen peroxide sensitivity of isc1Δ cells, increasing cell survival to levels similar to the observed for sch9Δ mutant: cell viability was approximately 27% in parental cells, 7% in isc1Δ cells and 35% in sch9Δ and isc1Δsch9Δ cells (figure 10). These results suggest that Sch9p contributes to increased oxidative stress in Isc1p-deficient cells. ! Figure-10. SCH9 disruption suppresses the oxidative stress sensitivity of isc1Δ cells. Yeast cells grown to exponential phase and exposed to 1.5mM H2O2 for 1 hour. Cell viability was expressed as the percentage of CFUs (treated cells vs. untreated cells). Data were expressed as mean values ± SD of at least three independent experiments. Values were compared by Student !s t-test. ***, p< 0.001; ****, p<0.0001. 0 10 20 30 40 50 (%) Viable cells isc1Δ sch9Δ sch9Δ isc1Δ BY4741 **** *** **** FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae 39 ! ! ! ! BY4741 isc1Δ! sch9Δ isc1 Δsch9Δ! IV-2. Characterization of mitochondrial function IV-2.1. SCH9 deletion relieves mitochondrial dysfunction of isc1Δ cells It was previously shown that sch9Δ cells have extended chronological lifespan and this is attributed, in part, to improved and better coupled mitochondrial respiration at early stages of growth, which ultimately preconditions yeast to better survive on the stationary phase (F; (Fabrizio et al., 2001; Wei et al., 2008, Pan et al., 2011). Since isc1Δ cells display severe mitochondrial dysfunction (Almeida et al., 2008, Barbosa et al., 2011), we evaluated if SCH9 disruption could improve mitochondrial fitness of this mutant strain. To address this hypothesis, we have analyzed different mitochondrial parameters, namely cell growth in medium containing glycerol, a non-fermentable carbon source, oxygen consumption and cytochrome c oxidase (COX) activity. To monitor respiratory capacity, we have firstly analyzed growth in glycerol, which requires functional mitochondria to metabolize it. For this purpose, yeast cells were grown to exponential phase, diluted to an OD600 = 0.1 and five-fold serial dilutions were performed in media containing either glucose or glycerol as carbon source. As expected, parental (BY4741) and sch9∆ cells were able to grow in glycerol whereas isc1∆ mutant cells were unable to grow in such conditions (Almeida et al., 2008, Barbosa et al., 2011). Importantly, it was observed that the growth defect of isc1Δ cells on glycerol medium was suppressed in the isc1Δsch9Δ double mutant (figure 11). Figure-11. Deletion of SCH9 restores respiratory capacity of isc1∆ cells. Yeast cells were grown to exponential phase, diluted to a OD600=0.1 and fivefold dilutions were plated in SC-medium containing glucose or glycerol as carbon source. Glucose Glycerol 46! FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae! ! parental cells upon shift to lactate-growing conditions (figure 17). Notably, isc1Δ cells presented enhanced alkaline phosphatase activity when compared to parental cells in similar conditions, suggesting that mitophagy is increased in the mutant strain. On the other hand, mitophagy induction was restored to values similar to parental cells in the isc1Δsch9Δ double mutant. These results suggest that Sch9p also mediates mitophagy induction in isc1Δ cells. Figure-17. Mitophagy induction is enhanced in isc1Δ cells by Sch9p-dependent mechanisms. Yeast cells were grown in SC-medium containing glucose to early exponential phase (GLUC) and then shifted to SC-medium containing lactate (LAC) for 48 hours. The mtPho8 assay was performed as described in Material and Methods. Data were expressed as mean values ± SD of at least three independent experiments. Values were compared by Student !s t-test. ****,p<0.0001. We have also monitored alteration in mitophagy induction during chronological aging (figure 18). In this case, cells were grown in SC-medium to the PDS phase and then transferred to water overtime. We observed that mitophagy is only significantly induced after a 7-day incubation period in such conditions in parental cells. However, mitophagy induction was already higher in isc1Δ cells at the PDS phase and became significantly induced in cells aged for 5 days, maintaining steady levels at day 7. Importantly, mitophagy induction reached a peak at day 3 in the double mutant isc1Δsch9Δ and then decreased to levels similar to those observed for parental cells and this was correlated with the period when Isc1p-deficient cells reached maximum induction. 0 50 100 150 200 mtPho8 activity (nmol nitrophenol. min -1. mg protein) GLUC LACT BY4741 isc1Δsch9Δisc1Δ sch9Δ ******** **** **** FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae 47 ! ! ! Figure -18. Mitophagy induction during chronological lifespan. S.cerevisiae, isc1Δ, sch9Δ and isc1Δsch9Δ cells were grown in SC-medium to the PDS phase, shifted to water and then kept throughout the assay. The mtPho8 activity was determined as described in Material and Methods. Data were expressed as mean values ± SD of at least three independent experiments. Values were compared by two-way ANOVA with Bonferroni correction.*,p<0.05;**,p<0.01;****,p<0.0001. 0357 0 100 200 300 400 mtPho8 activity (nmol nitrophenol. min -1. mg protein) BY4741 isc1Δ sch9Δ isc1Δsch9Δ *** **** **** **** **** **** **** (Days) 48! FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae! ! FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae 49 ! Chapter V Discussion 50! FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae! ! Sphingolipids, such as ceramide, sphingosine and sphingosine-1-phosphate, have emerged in the last decades as key bioactive molecules, with roles in differentiation, senescence, cell cycle arrest, apoptosis and stress responses (Hannun and Obeid, 2008). The ability of these lipids to modulate cell signalling through activation of downstream effectors (protein kinases and phosphatases) and modulation of protein trafficking and intracellular localization suggests complex roles for sphingolipids in cell metabolism and physiology (Cowart and Obeid, 2007). The link between sphingolipid signalling and redox regulation has been established in some studies. For example, sphingolipids regulate cellular redox homeostasis and the activity of sphingomyelinases and ceramidase can be modulated by ROS and glutathione levels (Won and Singh, 2006). In S. cerevisiae, Isc1p, an inositolphosphosphingolipid phospholipase C, is required for oxidative stress resistance, chronological lifespan and proper mitochondrial function (Almeida et al., 2008, Barbosa et al., 2011). However, the role of Isc1p in signal transduction during oxidative stress response and chronological aging is not fully understood. In this work, we explored the involvement of the Sch9p protein kinase on these signalling pathways. Some studies have reported that Sch9p regulates mitochondrial function and CLS (Pan and Shadel, 2009) and autophagy (Yorimitsu et al. 2007) by integrating nutrient signals from TORC1 with stress signals from sphingolipids (Huang et al, 2012). On this basis, we hypothesized that the activation of the TORC1-Sch9p axis could be implicated in isc1Δ phenotypes. Our lab has recently disclosed that TORC1 is activated in cells lacking Isc1p (Teixeira et al., unpublished results), thus we postulated that Sch9p may act downstream of TORC1 and contribute to mitochondrial dysfunction, hydrogen peroxide sensitivity and premature aging of isc1Δ cells. Here we provided evidence that Sch9p signalling is impaired in Isc1p-deficient cells. In fact, the deletion of SCH9 alleviates the oxidative stress sensitivity and mitochondrial dysfunctions of isc1Δ cells, which is compatible with Isc1p acting upstream of Sch9p. Such features are in agreement with previous studies showing that SCH9 deletion contributes to better mitochondrial coupling and fitness during active growth in yeast cells by Rim15p-dependent mechanisms, eliciting an adaptive response that preconditions yeast cells to better survive in the stationary phase and promote longevity (Pan et al., 2011). This is associated with increased translation of both nuclearand mtDNA-encoded subunits of the oxidative phosphorylation system (Bonawitz et al., 2007), which is consistent with higher oxygen consumption and COX activity observed in this study. In addition, SCH9 disruption improves oxidative stress resistance already at the exponential phase, which is further extended at later stages of growth (stationary phase), as reported by others (Wei et al., FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae 51 ! 2009). Importantly, the restoration of proper mitochondrial function and oxidative stress resistance mechanisms (Cta1p activity) in isc1Δsch9Δ cells was correlated with a decrease in ROS levels during the stationary phase. Since the deletion of SCH9 suppresses the shortened CLS of isc1Δ cells, this decrease probably contributes to lifespan extension in isc1Δsch9Δ cells (Teixeira et al., unpublished results). Our data suggest that the protein kinase Sch9p, the yeast homologue of mammalian Akt and pS6K proteins, acts downstream of TORC1 since the disruption of both TOR1 (Teixeira et al., unpublished results) and SCH9 (this study) abolishes isc1Δ phenotypes. However, such regulation may also occur by TORC1-independent mechanisms. Lipidomic analysis showed specific changes in sphingolipids that accompanied the premature ageing of Isc1p-deficient cells, including increased basal levels of phytosphingosine (PHS), raising the possibility that Sch9p may also be activated in response to sphingolipid metabolism, as reported by others (Huang et al., 2011). In addition to the phosphorylation of C-terminus by TORC1, Sch9p is phosphorylated in Thr570 in the activation loop by Pkh1p/Pkh2p protein kinases, in response to LCBs (Liu et al., 2005). Furthermore, the downregulation of sphingolipid synthesis enhances CLS and improves mitochondrial function and oxidative stress resistance through the modulation of the PHS-Pkh1/2p-Sch9p axis. On this basis, we hypothesize that Sch9p may also act by integrating sphingolipid signalling. Further studies are necessary to clarify this hypothesis. Studies performed in mammalian cells also support a functional connection between sphingolipid metabolism and the activation of Akt pathway and S6K. Qin et al. have recently demonstrated that fibroblasts with neutral sphingomyelinase 2 deficiency presented increased hyaluronan synthesis and secretion and this was correlated with the activation of Akt/mTOR pathway (increased phosphorylation of Akt) and p70S6K (Qin et al., 2012.). Autophagy is a degradative process for bulk proteins and damaged and/or unnecessary organelles, induced primarily in response to nutrient starvation (Mizushima and Klionsky, 2007). Recent studies have reported that autophagic defects have a functional impact on various aspects of mitochondrial functions, suggesting a critical role of autophagy in mitochondria functional integrity and maintenance (Zhang et al., 2007; Twig et al., 2008). The deletion mutants for essential autophagy genes exhibit various defects related to mitochondrial biology. For instance, Zhang Y et al. demonstrated that mutants defective in ATG genes (autophagy related genes) presented lower oxygen consumption rates, higher levels of ROS and they were more prone to accumulate dysfunctional mitochondria (Zhang et al., 2007). 52! FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae! ! TORC1 plays a major role in the regulation of autophagy and recent data have demonstrated that Sch9p is a negative regulator of the process (Yorimitsu et al., 2007). In this study, we show that Isc1p-deficient cells presented reduced autophagic flux, which could be on the etiology of mitochondrial dysfunctions exhibited by the mutant strain. Remarkably, SCH9 disruption reestablishes the autophagic flux in isc1Δ cells, which is consistent with the suppression of mitochondrial dysfunction, H2O2 hypersensitivity and premature aging in the double mutant. Mitochondria are dynamic structures that migrate throughout the cell, fuse and divide, and undergo regulated turnover by mitophagy. These highly coordinated processes are important on the regulation of mitochondrial function and cell physiology by allowing mitochondrial recruitment to critical subcellular compartments, mitochondrial communication, regulation of the mitochondrial shape and mitochondrial quality control. However, alterations in these quality control mechanisms affect overall cellular metabolism and cell fate. For instance, when mitochondrial dynamics is disrupted, mitochondrial dysfunction ensues and this is usually related with ageing and age-associated pathologies, such as cancer and neurodegenerative diseases (Lin and Beal, 2006; Anandatheerthavarada et al., 2003). On this basis, we have studied alterations in mitochondrial dynamics and mitophagy. In this study, we demonstrate that Isc1p-deficient cells have disrupted mitochondrial network, consistent with mitochondrial fragmentation, and that the deletion of SCH9 abolishes this phenotype. These results suggest that Sch9p is also implicated in the regulation of mitochondrial dynamics in isc1Δ cells, accounting for mitochondrial dysfunction displayed by this mutant strain. Mitophagy in yeast can be induced by transfering cells initially grown in a lactate medium for mitochondrial proliferation to nitrogen starvation medium with glucose (Kurihara et al., 2011). However, we already observed a significant induction of mitophagy in isc1Δ cells during growth on lactate medium, comparing with parental cells. Consequently, we have decided to study mitophagy only by shifting the cells from glucose to lactate medium. The increased mitophagy observed in isc1Δ cells is consistent with the fact that this mutant exhibits dysfunctional mitochondria. Thus, the induction of mitophagy may account as a clearance mechanism in an attempt to remove damaged mitochondria and possibly maintain a healthy population of organelles to fulfill cellular bioenergetics demands. However, some lines of evidence have demonstrated that mitophagy can also be activated in response to alterations in mitochondrial dynamics. For instance, Mao et al. have recently demonstrated that mutants cells lacking Dnm1p and Fis1p, which are involved in mitochondrial fission in yeast, present lower levels of mitophagy in inducible conditions, unraveling an important link between these control quality mechanisms (Mao et al., 2011). Thus, we also hypothesize that the increased FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae 53 ! mitophagy observed in isc1∆ cells could be attributed to increased mitochondrial fission since SCH9 deletion suppressed mitochondrial fragmentation in Isc1p-deficent cells and this was correlated with lower mitophagy induction in the double mutant isc1∆sch9∆. Further studies are necessary to substantiate this hypothesis. In order to obtain a more general framework, we have also analysed mitophagy during CLS. Aging can be defined as a multifactorial phenomenon characterized by a time-dependent decline in physiological function that results in a gradual structural and functional deterioration of biomolecules and impairment of stress resistance mechanisms. Mitochondria, as major sites of ROS production, contribute to the accumulation of damage to macromolecules, which in turn overwhelm the capacity of biological systems to repair themselves, resulting in an inevitable functional decline. To monitor the magnitude of these alterations during aging, we have firstly grown cells in SC-medium to the PDS phase and then transferred to water throughout. Such severe caloric restriction (CR) regime (incubation in water) was used to extend the monitoring of this process during CLS, since isc1Δ cells present premature aging and this regime results in the longest survival for wild-type yeast strains (Fabrizio and Longo, 2003). Our results suggest that Sch9p activation is also detrimental in the regulation of mitophagy in isc1Δ cells during aging, which is intimately related with the involvement of the protein kinase in mitochondrial dysfunction and impairment of mitochondrial dynamics in the mutant strain. Importantly, the diminished induction of mitophagy at late stages of growth (when mitophagy induction reaches a maximum in Isc1p-deficient cells) is associated with the extended CLS of isc1Δ cells upon deletion of SCH9 (Teixeira et al., unpublished results). 54! FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae! ! ! ! ! ! ! ! ! ! ! ! ! ! Chapter VI Conclusion FCUP Regulation of mitochondrial function by Isc1p and Sch9p in Saccharomyces cerevisiae 55 ! In summary, our data suggest that Isc1p functions upstream of Sch9p and implicate the protein kinase in mitochondrial dysfunction, premature ageing and oxidative stress sensitivity of Isc1p-defcient cells. In fact, SCH9 deletion restores mitochondrial function and proper morphology and the functional integrity of related biological processes, such as autophagy, mitophagy and oxidative stress response in isc1∆ cells. 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