title: Hyper- and hyporesponsive mutant forms of the Saccharomyces cerevisiae Ssy1 amino acid sensor.
abstract: The Saccharomyces cerevisiae integral membrane protein Ssy1p functions with Ssy5p and Ptr3p to sense extracellular amino acids. Signal transduction leads to processing and nuclear localization of Stp1p and Stp2p, transcriptional activators of many amino acid transporter genes. Ssy1p is structurally related to amino acid permeases, but unable to transport amino acids. We isolated SSY1 mutants that constitutively activate a target promoter. Dose-response analysis showed that the mutants are hyperresponsive, requiring less inducer to give strong signaling than does the wild type. Another mutant (Ssy1p(T639I)) turned out to be hyporesponsive, i.e., it signals only at high inducer concentration. In accordance with a transporter-like mechanism for Ssy1p function we suggest that the hyper- and hyporesponsive mutant forms differ from the wild-type sensor by being more and less inclined, respectively, to adopt an outward-facing, signaling conformation. Coordinate conformational dynamics of the sensor complex was supported by additive effects of combinations of constitutive SSY1, PTR3 and SSY5 alleles. Assuming structural similarity of Ssy1p to the distantly related bacterial leucine transporter LeuT(Aa), several activating substitutions were located near the substrate binding site while others were on the periphery of Ssy1p. We suggest analyses of transporter-like sensors as an approach to understand key features of transporters.
authors.affiliation: Carlsberg Laboratory, Copenhagen Valby, Denmark.
authors.forename: Peter
authors.initials: P
authors.lastname: Poulsen
authors.forename: Richard F
authors.initials: RF
authors.lastname: Gaber
authors.forename: Morten C
authors.initials: MC
authors.lastname: Kielland-Brandt
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D026921:Amino Acid Transport Systems, Neutral / Q000235:genetics; D000596:Amino Acids / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D016601:RNA-Binding Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism
chemical_list: C420623:AGP1 protein, S cerevisiae; D026921:Amino Acid Transport Systems, Neutral; D000596:Amino Acids; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D050505:Mutant Proteins; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; C120527:SSY1 protein, S cerevisiae; C074632:STP1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors