Studies of the rapamycin signaling pathway in the yeast Saccharomyces cerevisiae.

摘要

Rapamycin is a potent antifungal and immunosuppressant drug. Like T-cells, the yeast Saccharomyces cerevisiae treated with rapamycin arrests growth in the G1 phase of the cell cycle. The studies described herein have demonstrated a direct interaction between the complex of rapamycin and FKBP12 (drug receptor), and Tor2p (downstream protein in the rapamycin signaling pathway). This interaction is dependent on the presence of the drug and of a conserved Serine residue at position 1975 in Tor2p. Mutations at this locus inhibit the interaction with the complex of rapamycin-FKBP12, and thus confer dominant rapamycin resistance. Two independent mutations, N2284K and D2298E, were introduced into the PI 3/protein kinase-related domain of wild type TOR2 (S1975) and a dominant rapamycin resistant TOR2 (S1975R). Overexpression of the kinase-related domain mutants had a dominant negative effect on growth. In the presence of rapamycin, TOR2 S1975R N2284K and S1975R D2298E became rapamycin sensitive, which suggests that Tor2p requires an intact kinase domain in order to perform its G1 function. Further analysis of these mutants indicated that Tor2p essential function also requires an intact kinase domain. Promoter studies of a TOR2 homologue, TOR1, and TOR2 itself, identified the transcription initiation start site for both genes, and further experiments proved that both promoters have very weak activity in vivo. A genetic screen for additional genes involved in rapamycin sensitivity identified TAT1, which encodes an amino acid permease with high affinity for tyrosine and low affinity for tryptophan. Wild-type TAT1 is able to confer weak rapamycin resistance when present in only one extra copy per cell, and the resistance increases with the gene copy number. Tyrosine transport assays indicated that rapamycin had no specific inhibitory effect on tyrosine uptake.