Genetic analysis of the signalling pathway activated by external amino acids in Saccharomyces cerevisiae

The permease‐like amino acid sensor Ssy1p of Saccharomyces cerevisiae is required for transcriptional induction, in response to external amino acids, of several genes encoding peptide and amino acid permeases. Among them is AGP1 encoding a low‐affinity, broad‐specificity amino acid permease important for the utilization of amino acids as a nitrogen source. We report here data from experiments aimed at identifying components of the signalling pathway activated by Ssy1p. Overproduction of the large amino‐terminal tail of Ssy1p interferes negatively with the induction of AGP1 in wild‐type cells. Furthermore, overproduction of this domain can relieve growth defects of a ssy1 null strain, indicating that the N‐terminal tail of Ssy1p is an important functional element of the pathway. Consistent with a role for Ssy1p in the recognition of amino acids, a mutant form of the protein with a Thr to Ile substitution in the eighth predicted transmembrane domain is competent for the induction of AGP1 by leucine but not by other amino acids. In a screen for other mutants defective in the Ssy1p pathway, we confirmed that PTR3 and SSY5 encode additional factors essential for AGP1 expression in response to multiple amino acids. Data obtained by overproducing Ptr3p and Ssy5p in ssy1Δ , ptr3Δ and ssy5Δ mutants suggest that Ptr3p acts downstream from Ssy1p and Ssy5p downstream from Ptr3p in the transduction pathway. Furthermore, two‐hybrid experiments indicated that Ptr3p interacts with Ssy5p and that Ptr3p can self‐associate. Finally, the Cys‐6‐Zn 2 transcription factor Uga35p/Dal81p required for the induction of AGP1 is also essential for the expression of two other genes under Ssy1p–Ptr3p–Ssy5p control, namely BAP2 and PTR2 , suggesting that the protein is yet another component of the amino acid signalling pathway.