Figure 2.

TOR1 kinase domain is essential for TOR1 function and rapamycin resistance, and overexpression of TOR1 kinase-inactive mutant inhibits growth. (A) A tor1 Δsrk1 yeast strain (CAY7) was transformed with 2μ plasmids expressing wild-type TOR1, the S1972I rapamycin-resistant TOR1 mutant, the D2275A kinase-inactive TOR1 mutant, or the S1972I/D2275A double-mutant TOR1 protein. Plasmids expressing TOR1 function complement the tor1 srk1 conditional lethal phenotype and rescue cell viability at 39°C, whereas plasmids lacking TOR1 function do not. Cells were grown for 72 h at 30 and 39°C. (B) Wild-type strain JK9–3da was transformed with 2μ plasmids expressing wild-type TOR1, the kinase-inactive D2275A mutant TOR1, four independent rapamycin-resistant TOR1 mutant isolates (S1972I), and two independent isolates of the S1972I/D2275A TOR1 double mutant. Cells were grown on medium containing 1 μg/ml rapamycin for 72 h at 30°C. (C) Yeast strain CAY6 expressing wild-type TOR1 (TOR1), the TOR1 kinase-inactive mutant (TOR1 D2275A), the TOR1 rapamycin-resistant mutant (TOR1 S1972I), or the TOR1 kinase-inactive rapamycin-resistant double mutant (TOR1 S1972I/D2275A) from the galactose-inducible GAL1 promoter were grown on glucose or galactose medium lacking tryptophan for 96 h at 30°C.