Constitutive Signal Transduction by Mutant Ssy5p and Ptr3p Components of the SPS Amino Acid Sensor System in Saccharomyces cerevisiae

Abstract

Amino acids in the environment of Saccharomyces cerevisiae can transcriptionally activate a third of the amino acid permease genes through a signal that originates from the interaction between the extracellular amino acids and an integral plasma membrane protein, Ssy1p. Two plasma membrane-associated proteins, Ptr3p and Ssy5p, participate in the sensing, which results in cleavage of the transcription factors Stp1p and Stp2p, removing 10 kDa of the N terminus of each of them. This confers the transcription factors with the ability to gain access to the nucleus and activate transcription of amino acid permease genes. To extend our understanding of the role of Ptr3p and Ssy5p in this amino acid sensing process, we have isolated constitutive gain-of-function mutants in these two components by using a genetic screening in which potassium uptake is made dependent on amino acid signaling. Mutants which exhibit inducer-independent processing of Stp1p and activation of the amino acid permease gene AGP1 were obtained. For each component of the SPS complex, constitutive signaling by a mutant allele depended on the presence of wild-type alleles of the other two components. Despite the signaling in the absence of inducer, the processing of Stp1p was more complete in the presence of inducer. Dose response assays showed that the median effective concentration for Stp1p processing in the mutant cells was decreased; i.e., a lower inducer concentration is needed for signaling in the mutant cells. These results suggest that the three sensor components interact intimately in a complex rather than in separate reactions and support the notion that the three components function as a complex.

Mapping of an Internal Protease Cleavage Site in the Ssy5p Component of the Amino Acid Sensor of Saccharomyces cerevisiae and Functional Characterization of the Resulting Pro- and Protease Domains by Gain-of-Function Genetics

“…␤-Galactosidase activity in these transformants grown in the absence of leucine was increased over that of control cells expressing a plasmid-borne wild-type SSY5 allele. These results (not shown) are similar to those found for the previously characterized constitutive mutant SSY5-6 (28). Like this mutant, the new mutants were also dependent on the presence of Ssy1p and Ptr3p for constitutive signaling (data not shown).…”

“…This suggests that Ssy5p is responsible for the proteolytic removal of the ϳ10-kDa N-terminal fragment of each of the transcription factors Stp1p and Stp2p, resulting in their migration from the cytoplasm/plasma membrane to the nucleus (1, 2, 3, 4). Signaling has been measured by the activation of target promoters, such as the BAP2 promoter (12,26) or the AGP1 promoter (21), and by quantifying the proteolytic processing of Stp1p processing (27,28).…”

“…SSY5 mutants were characterized for the dose-response relationships between amino acid exposure and Stp1p processing as described (27,28).…”

Differential Regulation of Transcription Factors Stp1 and Stp2 in the Ssy1-Ptr3-Ssy5 Amino Acid Sensing Pathway

“…Ssy5 has been proposed to form a plasma membrane-localized sensor complex with Ssy1 and Ptr3 (7,11,20,31). It has also been reported that Ssy1 is required for efficient plasma membrane localization of Stp1 using the Sos recruitment system (18).…”

“…To analyze post-translational modifications of Stp1 and Stp2, we constructed C-terminal HA-tagged constructs of Stp1 and Stp2, which were found to be able to fully restore amino acid-induced AGP1-lacZ expression to stp1⌬ stp2⌬ mutant cells, indicating that they are functional (data not shown). The AGP1-lacZ fusion gene has been used extensively as a reporter in this pathway (2,11,30,31). Previously, both full-length and processed Stp1 have been reported to be unstable, with a half-life (t1 ⁄ 2 ) of Ͻ10 min for full-length Stp1 (18,24).…”