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 re quired for transcriptional induction, in response to external amino acids, of several genes encoding peptide and amino acid permeases. Among them is A GP1 encoding a low-affinity, broad-specificity amino acid permease importan t for the utilization of amino acids as a nitrogen source. We report here d ata from experiments aimed at identifying components of the signalling path way 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 import ant 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 lie substitution in the eighth predicted transmembrane domain is competent for the induction of AGP1 by leucine but not by other amino acids. In a scr een for other mutants defective in the Ssy1p pathway, we confirmed that PTR 3 and SSY5 encode additional factors essential for AGP1 expression in respo nse to multiple amino acids. Data obtained by overproducing Ptr3p and Ssy5p in ssy1 Delta, ptr3 Delta and ssy5 Delta mutants suggest that Ptr3p acts d ownstream from Ssy1p and Ssy5p downstream from Ptr3p in the transduction pa thway. Furthermore, two-hybrid experiments indicated that Ptr3p interacts w ith Ssy5p and that Ptr3p can self-associate. Finally, the Cys-6-Zn-2 transc ription factor Uga35p/DaI81p required for the induction of AGP1 is also ess ential for the expression of two other genes under Ssy1p-Ptr3p-Ssy5p contro l, namely BAP2 and PTR2, suggesting that the protein is yet another compone nt of the amino acid signalling pathway.