Eckert-Boulet, Nadine

Gæren Saccharomyces cerevisiae har udviklet komplekse regulatoriske systemer til at kontrollere ekspression af de proteiner, der importerer næringsstoffer, således at disse kun bliver produceret, når der er brug for dem. Dette er tilfældet for hexose-transportører samt aminosyre-transportører (disse bliver også kaldt amino acid permeases (AAPs)). Deres ekspression induceres på det transkriptionelle niveau efter at ekstracellulære næringsstoffer, henholdsvis glukose og aminosyrer, bliver detekteret af sensor-proteiner, som sidder i plasmamembranen. Aminosyrer bliver detekteres af den såkaldte SPS (Ssy1p-Ptr3p-Ssy5p) sensor, og et signal genereres og bliver transmitteret via de homologe transkriptionsfaktorer Stp1p og Stp2p. Stp1p og Stp2p undergår endoproteolytisk kløvning i cytoplasma og migrerer derefter til cellekernen, hvor de binder til promoter-DNA ved flere AAP gener, hvilket medfører transkriptionel induktion. For at kunne bekræfte eller identificere nye gener som reguleres via SPS-systemet, blev der udført global transkriptionel profilering af vildtype stammer samt af stammer deleterede for enten SSY1 eller STP1 og STP2, både med og uden aminosyren L-citrullin i mediet. L-citrulline kan ikke optages af disse stammer, da de mangler GAP1 (General Amino acid Permease). Det blev bekræftet, at AAP generne TAT1, BAP2, BAP3 og PTR2 er under kontrol af aminosyre induktionsvejen, mens AGP2 blev identificeret som et nyt gen under kontrol af denne mekanisme. Den globale transkriptionsanalyse viste at 46 gener induceres af L-citrullin, afhængigt af Ssy1p og Stp1p/Stp2p. Sekvenssammenligning af promoterregionerne muliggjorde en mere præcis definition af den tidligere beskrevet Upstream Activating Sequence (UASaa). Udover effekten på AAP gener, viste ssy1 og stp1 stp2 mutanter mange andre transkriptionelle fænotyper, såsom øget ekspression af gener under kontrol af Nitrogen Catabolite Repression og gener involverede i stress respons. F-box proteinet Grr1p, som også spiller en rolle i regulering af cellecyklus og glukose induktion af heksose transportørgener, er nødvendig for at aminosyre induktion kan finde sted. Hel-genom transkriptionsanalyser af en vildtype stamme og en grr1 stamme med og uden L-citrullin blev udført. Dataanalyse viste at aminosyre induktion af AAP generne AGP1, BAP2, BAP3, DIP5, TAT1, og GNP1 er helt afhængig af tilstedeværelsen af GRR1. Sammenligningen af de to stammers transkriptionelle profiler viste at, når GRR1 er slået ud, transkription af mange gener til enzymer i den centrale karbon metabolisme øges. Promoteranalyse viste at mange af de gener med øget transkription har mulige Mig1p og/eller Msn2p-Msn4p bindingelementer. Den øgede ekspression af glukose-represserede gener i grr1 stammen kan være en indirekte konsekvens af den begrænsede glukose optagelse som forventes i denne mutant på grund af formindsket ekspression af flere hexose transportører. I et forsøg på at identificere nye komponenter i SPS-signaltransduktionsmekanismen, blev en transposon samling screenet for mutanter med (konstitutive) transkriptionel induktion af AAP gener, det vil sige, mutanter, i hvilke signaleringsvejen er aktiv selv uden aminosyre i mediet. Flere transposoner blev fundet i RTS1 locus. RTS1 koder en af de to regulatoriske enheder af protein fosfatase 2A, som er kendt i gær. RTS1 blev slået ud, og dette medførte konstitutiv aktivering af AGP1 og BAP2 promotorer. Dette var afhængigt af SSY1, PTR3, SSY5, GRR1 eller STP1, STP2 og deres homolog STP3. Den forøgede transkription fra AGP1 og BAP2 promotorer i rts1 celler ser ud til at skyldes forøget processering af Stp1p. Med dette arbejde blev der identificeret nye gener som reguleres via SPS-signalnetværket og konsensussekvensen af UASaa involveret i aminosyre induktion blev defineret mere præcist. Det er blevet bekræftet at Grr1p er involveret i denne vej og effekterne af grr1 mutationen blev undersøgt på hel-genom transkriptionelt niveau. Derudover, blev der identificeret en ny komponent af signalnetværket, som er protein fosfatase 2A sammen med sin regulatoriske enhed Rts1p, og som udfører en negativ regulering af vejen.The yeast Saccharomyces cerevisiae has developed complex regulatory systems to control expression of nutrient transporters so that these are only produced when needed. This is the case for hexose transporters and amino acid transporters (the latter are known as amino acid permeases (AAPs)). Their expression is induced at the transcriptional level following detection of extracellular nutrients, glucose and amino acids, respectively, by sensor proteins located at the plasma membrane. Amino acids are sensed by the SPS (Ssy1p-Ptr3p-Ssy5p) sensor. The resulting signal is transmitted to the homologous transcription factors, Stp1p and Stp2p, which undergo endoproteolytic processing in the cytoplasm and migrate to the nucleus, where they bind an Upstream Activating Sequence (UASaa) present in the promoter DNA of several AAP genes, thereby inducing transcription. In order to confirm or identify targets of this pathway, whole-genome transcription profiles of wild-type strains and of strains deleted in either SSY1 or STP1 and STP2 were compared using DNA microarrays, in the absence and presence of the amino acid Lcitrulline. L-citrulline cannot be taken up by the strains used, since they are devoid of the General Amino acid Permease Gap1p. The AAP genes TAT1, BAP2, BAP3 and PTR2 were confirmed to be under the control of the amino acid induction pathway, while AGP2 was identified as a new AAP gene target. Global transcription analysis showed that 46 genes were induced by L-citrulline, in a manner dependent on SSY1 and STP1/STP2. Alignment of the promoter sequences allowed a more precise definition of the consensus sequence of the UASaa identified previously. Besides the effects on AAP genes, ssy1∆ and stp1∆ stp2∆ mutants exhibited a number of other transcriptional phenotypes, such as increased expression of genes subject to Nitrogen Catabolite Repression and genes involved in stress response. The F-box protein Grr1p, known for its role in cell cycle regulation and glucose induction of the hexose transporter genes, is required for amino acid induction to occur. Genomewide transcription analysis of a wild-type strain and of a grr1∆ strain, in the absence and presence of L-citrulline, were performed using DNA microarrays. L-citrulline cannot be taken up in the experimental conditions used. Data analysis showed that amino acid induction of the AAP genes AGP1, BAP2, BAP3, DIP5, TAT1, and GNP1 is completely dependent on the presence of GRR1. Comparison of the transcriptional profiles of the two strains in the absence of inducer revealed that GRR1 disruption leads to increased transcription of numerous genes encoding enzymes of the central carbon metabolism. In addition, promoter analysis showed that many of the genes with increased transcription display Mig1p- and/or Msn2p/Msn4p-binding sites. Increased expression of glucoserepressed genes in the grr1∆ strain may be an indirect consequence of the reduced glucose uptake expected from reduced expression of several hexose transporter genes in such a strain. In an attempt to identify novel components of the SPS-mediated pathway, a transposon mutant library was screened for mutants exhibiting (constitutive) transcriptional induction of the AAP genes, i.e. mutants in which the pathway is active even in the absence of inducer. Several transposons were found at the RTS1 locus. RTS1 encodes one of the two regulatory subunits of protein phosphatase 2A known in yeast. Deletion of RTS1 indeed resulted in constitutive activation of the amino acid-inducible AGP1 and BAP2 promoters, in a manner that was dependent on SSY1, PTR3, SSY5, GRR1 or STP1, STP2 and their homologue STP3. Increased transcription from the AGP1 and BAP2 promoters in rts1∆ cells appeared to occur via increased Stp1p processing. Through this work, new targets of the SPS-mediated pathway have been identified and the consensus sequence of the UASaa involved in amino acid induction has been defined more precisely. The involvement of Grr1p has been confirmed and the effects of a GRR1 deletion at the whole-genome transcriptional levels investigated. In addition, a new component of the pathway, namely protein phosphatase 2A associated with its regulatory subunit Rts1p, has been identified as a down-regulating factor

Online Research Database In Technology

Investigation of nutrient sensing in the yeast Saccharomyces cerevisiae

http://orbit.dtu.dk/ws/files/5063387/thesis%20-%20nadine%20eckert%20boulet.pdf

Investigation of nutrient sensing in the yeast Saccharomyces cerevisiae

Abstract

Similar works

Full text

Available Versions

Online Research Database In Technology