Investigation of nutrient sensing in the yeast Saccharomyces cerevisiae

Nadine Eckert-Boulet

    Research output: Book/ReportPh.D. thesis

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    Abstract

    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.
    Original languageEnglish
    Place of PublicationKgs. Lyngby
    PublisherTechnical University of Denmark
    Number of pages105
    ISBN (Print)87-91494-43-5
    Publication statusPublished - Mar 2006

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