The target of rapamycin (TOR) kinase is an important regulator of growth in eukaryotic cells. In budding yeast, Tor1p and Tor2p function as part of two distinct protein complexes, TORC1 and TORC2, where TORC1 is specifically inhibited by the antibiotic rapamycin. Significant insight into TORC1 function has been obtained using rapamycin as a specific small molecule inhibitor of TOR activity. Here we show that caffeine acts as a distinct and novel small molecule inhibitor of TORC1: (i) deleting components specific to TORC1 but not TORC2 renders cells hypersensitive to caffeine; (ii) rapamycin and caffeine display remarkably similar effects on global gene expression; and (iii) mutations were isolated in Tor1p, a component specific to TORC1, that confers significant caffeine resistance both in vivo and in vitro. Strongest resistance requires two simultaneous mutations in TOR1, the first at either one of two highly conserved positions within the FRB (rapamycin binding) domain and a second at a highly conserved position within the ATP binding pocket of the kinase domain. Biochemical and genetic analyses of these mutant forms of Tor1p support a model wherein functional interactions between the FRB and kinase domains, as well as between the FRB domain and the TORC1 component Kog1p, regulate TOR activity as well as contribute to the mechanism of caffeine resistance.
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