Convergence of phosphate-specific and general nutrient sensing mechanisms in Saccharomyces cerevisiae.

Anorganisch fosfaat (Pi) is een essentieel nutriënt voor alle levende or ganismen aangezien het noodzakelijk is voor de synthese van ATP, de bela ngrijkste energiebron voor de cel. De plasmamembraan is echter impermeab el voor fosfaatmolecules en daarom zijn Pi transporters noodzakelijk voo r het transporteren van Pi vanuit de extracellulaire omgeving naar het c ytoplasma. De gist Saccharomyces cerevisiae heeft vijf Pi-transporter s: Pho84, Pho87, Pho89, Pho90 en Pho91. De eerste vier bevinden zich aan de plasma membraan, terwijl Pho91 zich in de vacuolaire membraan bevind t waar het Pi transporteert vanuit het cytoplasma naar de vacuole. De vi er plasmamembraan Pi-transporters omvatten twee verschillende Pi-transpo rtsystemen: een lage-affiniteitssysteem (Pho87 en Pho90) en een hoge-aff initeitssysteem (Pho84 en Pho89). De expressie van PHO87 en PHO90& nbsp; is constitutief en beide Pi-transporters mediëren Pi-transport wan neer genoeg extracellulair Pi aanwezig is. Onder Pi-limiterende conditie s wordt de transcriptie van het hoge-affiniteitssysteem geïnduceerd, ter wijl de activiteit van de lage affiniteit Pi-transporters wordt geïnhibe erd. Dus, waneer Pi limiterend wordt, nemen Pho84 en Pho89 de Pi-transpo rtfunctie over van Pho87 en Pho90 wat leidt tot verhoogde Pi-acquisitie. Dit proces wordt geregeld via activatie van de PHO-signaalweg. Pi-limit atie leidt tot activatie van Pho81. Dit proteïne inhibeert de activiteit van het Pho85-Pho80 kinase, wat resulteert in nucleaire translokatie va n de Pho4 transcriptiefactor. Nucleair Pho4 induceert de transcriptie va n de zogenaamde PHO-genen en deze genen omvatten naast PHO84 en PHO89 ook SPL2, de inhibitor van het lage-affiniteitssysteem. Wanneer v oldoende Pi aanwezig is, leidt dit tot inhibitie van Pho81 en vervolgens tot een actief Pho85-Pho80 kinase. Dit kinase hyperfosforyleert dan Pho 4, wat leidt tot zijn nucleaire exclusie en tot repressie van de PHO-gen en. In dit werk werd er onderzocht of andere nutriënten zoals stikstof of koolstof de Pi-respons van gist konden beïnvloeden. We tonen aan dat de lage-affiniteit Pi-transporters vacuolaire translokatie ondergaan wa nneer Pi limiterend wordt, maar dat de regulatie van dit proces verschil lend is voor Pho87 en Pho90. Terwijl dit proces voor Pho87 volledig afha nkelijk is van Spl2, leidt deletie van SPL2 niet tot stabilisatie van Pho90 tijdens deze condities. Wanneer andere nutriënten limiterend word en (koolstof/stikstof), ondergaan beide Pi-transporters vacuolaire trans lokatie onafhankelijk van Spl2. Interessant was wel dat het SPX-domein v an beide transporters noodzakelijk is voor dit proces onder alle nutriëntco ndities. Er wordt dus voldoende bewijs geleverd voor het feit dat Pho87 en Pho90 niet-redundante Pi-transporters zijn en dat andere nutritionele condities naast Pi ook de Pi-transportcapaciteit van een gistcel kunnen beïnvloeden. Vervolgens werd de lokalisatie van de Pho4 transcriptiefactor onderzocht onder verschillende nutriëntcondities. Er was al aangetoond dat Pho4 nu cleaire translokatie ondergaat wanneer Pi limiterend wordt. Hier tonen w e aan dat wanneer glucose verwijderd wordt, dit leidt tot nucleaire excl usie van Pho4 en dat dit proces gereguleerd wordt door de glucose-repres sie signaalweg. Additioneel demonstreren we dat inductie van stikstoflim itatie via inhibitie van TORC1 leidt tot nucleaire translokatie van Pho4 , zelfs wanneer voldoende Pi aanwezig is en dit proces kan gemimickt wor den via deletie van SCH9, een belangrijk substraat van TORC1. Interes sant is wel dat er al was aangetoond dat een gecombineerde deletie van S CH9 en PHO85 lethaal is voor gistcellen en nu blijkt dat dit fenot ype veroorzaakt wordt door deregulatie van de Pho4 transcriptiefactor. S ch9 en Pho85-Pho80 regelen dus in parallel de lokalisatie van Pho4 onder verschillende nutriëntcondities en deregulatie van dit proces door geco mbineerde deletie van SCH9 en PHO85 resulteert in een synthetisch lethaal fenotype. Finaal werd er onderzocht of bepaalde extracellulaire ionen een effect h ebben op de groei van gistmutanten die niet in staat zijn de PHO-signaal weg te activeren. Hier tonen we aan dat zo n giststammen geen groei vert onen in aanwezigheid van hoge concentraties aan Fe2+ en bij alkalische p H. Meer nog, voor groei in aanwezigheid van hoge concentraties aan Ca2+ is alleen maar activatie van de PHO-signaalweg vereist wanneer Ca2+-sign alering afwezig is. Al deze Pho4-afhankelijke fenotypes worden echter op geheven wanneer extra anorganisch Pi wordt toegediend aan het medium, wa t aantoont dat al deze condities leiden tot een daling in extracellulair e Pi-beschikbaarheid. Er moet dus rekening gehouden worden met het feit dat bepaalde ionen nutriënt-geïnduceerde signaalwegen kunnen activeren v ia het beïnvloeden van de beschikbaarheid van het specifiek nutriënt. &n bsp;nrpages: 212status: publishe

Molecular mechanisms linking the evolutionary conserved TORC1-Sch9 nutrient signalling branch to lifespan regulation in Saccharomyces cerevisiae

The knowledge on the molecular aspects regulating ageing in eukaryotic organisms has benefitted greatly from studies using the budding yeast Saccharomyces cerevisiae. Indeed, many aspects involved in the control of lifespan appear to be well conserved among species. Of these, the lifespan-extending effects of calorie restriction (CR) and downregulation of nutrient signalling through the target of rapamycin (TOR) pathway are prime examples. Here, we present an overview on the molecular mechanisms by which these interventions mediate lifespan extension in yeast. Several models have been proposed in the literature, which should be seen as complementary, instead of contradictory. Results indicate that CR mediates a large amount of its effect by downregulating signalling through the TORC1-Sch9 branch. In addition, we note that Sch9 is more than solely a downstream effector of TORC1, and documented connections with sphingolipid metabolism may be particularly interesting for future research on ageing mechanisms. As Sch9 comprises the yeast orthologue of the mammalian PKB/Akt and S6K1 kinases, future studies in yeast may continue to serve as an attractive model to elucidate conserved mechanisms involved in ageing and age-related diseases in humans.status: publishe

Life in the midst of scarcity: adaptations to nutrient availability in Saccharomyces cerevisiae

Cells of all living organisms contain complex signal transduction networks to ensure that a wide range of physiological properties are properly adapted to the environmental conditions. The fundamental concepts and individual building blocks of these signalling networks are generally well-conserved from yeast to man; yet, the central role that growth factors and hormones play in the regulation of signalling cascades in higher eukaryotes is executed by nutrients in yeast. Several nutrient-controlled pathways, which regulate cell growth and proliferation, metabolism and stress resistance, have been defined in yeast. These pathways are integrated into a signalling network, which ensures that yeast cells enter a quiescent, resting phase (G0) to survive periods of nutrient scarceness and that they rapidly resume growth and cell proliferation when nutrient conditions become favourable again. A series of well-conserved nutrient-sensory protein kinases perform key roles in this signalling network: i.e. Snf1, PKA, Tor1 and Tor2, Sch9 and Pho85–Pho80. In this review, we provide a comprehensive overview on the current understanding of the signalling processes mediated via these kinases with a particular focus on how these individual pathways converge to signalling networks that ultimately ensure the dynamic translation of extracellular nutrient signals into appropriate physiological responses

Development and validation of a humanized yeast model for Abeta42-induced cytotoxicity

Alzheimer’s Disease (AD) is the most common neurodegenerative disorder. Today, more than 25 million people are suffering from AD, a number that has been estimated to double every 20 years. Brains from AD patients have two major hallmarks. They contain intracellular deposits of hyperphosphorylated protein tau, known as neurofibrillary tangles, as well as extracellular deposits of Aβ peptides, known as amyloid plaques. The latter are derived from cleavage of the amyloid precursor protein by the β- and gamma-secretases. Despite enormous efforts, the exact molecular mechanisms underlying the pathology and neuronal loss in AD brain are still not known. According to a current hypothesis, the accumulation of intracellular oligomeric complexes of Aβ-peptides induces cytotoxicity and cellular stress, which in turn activates stress-sensitive kinases that hyper-phosphorylate and induce aggregation of protein tau. In our project, we aim to develop a humanized yeast model to investigate Aβ-induced toxicity and its causal effect on the behavior of protein tau in the unicellular eukaryote Saccharomyces cerevisiae or baker’s yeast, a strategy already successfully applied to decipher the toxicity mechanisms for other proteins triggering neurodegeneration in e.g. Parkinson’s and Huntington’s disease. Preliminary data shows that the proteasome plays an important role in clearing Aβ42, as severe growth defects are observed in proteasomally-impaired strains.status: publishe

Lipid droplet–mediated ER homeostasis regulates autophagy and cell survival during starvation

Lipid droplets (LDs) are conserved organelles for intracellular neutral lipid storage. Recent studies suggest that LDs function as direct lipid sources for autophagy, a central catabolic process in homeostasis and stress response. Here, we demonstrate that LDs are dispensable as a membrane source for autophagy, but fulfill critical functions for endoplasmic reticulum (ER) homeostasis linked to autophagy regulation. In the absence of LDs, yeast cells display alterations in their phospholipid composition and fail to buffer de novo fatty acid (FA) synthesis causing chronic stress and morphologic changes in the ER. These defects compromise regulation of autophagy, including formation of multiple aberrant Atg8 puncta and drastically impaired autophagosome biogenesis, leading to severe defects in nutrient stress survival. Importantly, metabolically corrected phospholipid composition and improved FA resistance of LD-deficient cells cure autophagy and cell survival. Together, our findings provide novel insight into the complex interrelation between LD-mediated lipid homeostasis and the regulation of autophagy potentially relevant for neurodegenerative and metabolic diseases

Development and validation of a humanized yeast model for Abeta42-induced cytotoxicity

Alzheimer’s Disease (AD) is the most common neurodegenerative disorder. Brains from AD patients have two major hallmarks. They contain intracellular deposits of hyperphosphorylated protein tau, known as neurofibrillary tangles, as well as extracellular deposits of Aβ peptides, known as amyloid plaques. The latter are derived from cleavage of the amyloid precursor protein (APP) by - and -secretases. Despite enormous efforts, the exact molecular mechanisms underlying the pathology and neuronal loss in AD brain are still not known. In our project, we aim to develop a humanized yeast model to investigate Aβ-induced toxicity and its causal effect on the behavior of protein tau in the unicellular eukaryote Saccharomyces cerevisiae. While other groups are focusing on the use of Aβ fusion proteins, we believe that while this approach will be able to provide answers for several questions, the addition of relatively large tags will obscure the true physiological behavior of small peptides. Therefore, we are developing a model that is physiologically more relevant, starting from the expression of either full length APP or the C99 fragment. Alongside these proteins we express functional β- and/or γ-secretases, so that cleavage of APP or C99 will result in the production of native Aβ-peptides. In our opinion, this model will provide a more truthful and representative way of researching amyloid beta-induced cytotoxicity. Preliminary data have already shown that co-expression of APP and both secretases causes a significant growth decrease in our yeast cells, in comparison to cells expressing only APP, or only β- and γ-secretase.status: publishe

Life in the midst of scarcity: adaptations to nutrient availability in Saccharomyces cerevisiae

Cells of all living organisms contain complex signal transduction networks to ensure that a wide range of physiological properties are properly adapted to the environmental conditions. The fundamental concepts and individual building blocks of these signalling networks are generally well-conserved from yeast to man; yet, the central role that growth factors and hormones play in the regulation of signalling cascades in higher eukaryotes is executed by nutrients in yeast. Several nutrient-controlled pathways, which regulate cell growth and proliferation, metabolism and stress resistance, have been defined in yeast. These pathways are integrated into a signalling network, which ensures that yeast cells enter a quiescent, resting phase (G0) to survive periods of nutrient scarceness and that they rapidly resume growth and cell proliferation when nutrient conditions become favourable again. A series of well-conserved nutrient-sensory protein kinases perform key roles in this signalling network: i.e. Snf1, PKA, Tor1 and Tor2, Sch9 and Pho85-Pho80. In this review, we provide a comprehensive overview on the current understanding of the signalling processes mediated via these kinases with a particular focus on how these individual pathways converge to signalling networks that ultimately ensure the dynamic translation of extracellular nutrient signals into appropriate physiological responses.status: publishe

Ca2+ homeostasis in the budding yeast Saccharomyces cerevisiae: Impact of ER/Golgi Ca2+ storage

Yeast has proven to be a powerful tool to elucidate the molecular aspects of several biological processes in higher eukaryotes. As in mammalian cells, yeast intracellular Ca(2+) signalling is crucial for a myriad of biological processes. Yeast cells also bear homologs of the major components of the Ca(2+) signalling toolkit in mammalian cells, including channels, co-transporters and pumps. Using yeast single- and multiple-gene deletion strains of various plasma membrane and organellar Ca(2+) transporters, combined with manipulations to estimate intracellular Ca(2+) storage, we evaluated the contribution of individual transport systems to intracellular Ca(2+) homeostasis. Yeast strains lacking Pmr1 and/or Cod1, two ion pumps implicated in ER/Golgi Ca(2+) homeostasis, displayed a fragmented vacuolar phenotype and showed increased vacuolar Ca(2+) uptake and Ca(2+) influx across the plasma membrane. In the pmr1Δ strain, these effects were insensitive to calcineurin activity, independent of Cch1/Mid1 Ca(2+) channels and Pmc1 but required Vcx1. By contrast, in the cod1Δ strain increased vacuolar Ca(2+) uptake was not affected by Vcx1 deletion but was largely dependent on Pmc1 activity. Our analysis further corroborates the distinct roles of Vcx1 and Pmc1 in vacuolar Ca(2+) uptake and point to the existence of not-yet identified Ca(2+) influx pathways.publisher: Elsevier articletitle: Ca2+ homeostasis in the budding yeast Saccharomyces cerevisiae: Impact of ER/Golgi Ca2+ storage journaltitle: Cell Calcium articlelink: http://dx.doi.org/10.1016/j.ceca.2015.05.004 content_type: article copyright: Copyright © 2015 Elsevier Ltd. All rights reserved.status: publishe

Phosphorylation, lipid raft interaction and traffic of α-synuclein in a yeast model for Parkinson

Parkinson's disease is a neurodegenerative disorder characterized by the formation of Lewy bodies containing aggregated α-synuclein. We used a yeast model to screen for deletion mutants with mislocalization and enhanced inclusion formation of α-synuclein. Many of the mutants were affected in functions related to vesicular traffic but especially mutants in endocytosis and vacuolar degradation combined inclusion formation with enhanced α-synuclein-mediated toxicity. The screening also allowed for identification of casein kinases responsible for α-synuclein phosphorylation at the plasma membrane as well as transacetylases that modulate the α-synuclein membrane interaction. In addition, α-synuclein was found to associate with lipid rafts, a phenomenon dependent on the ergosterol content. Together, our data suggest that toxicity of α-synuclein in yeast is at least in part associated with endocytosis of the protein, vesicular recycling back to the plasma membrane and vacuolar fusion defects, each contributing to the obstruction of different vesicular trafficking routes