FIGURE 3.

Rapamycin-insensitive proliferation is dependent on the presence of TORC1 components. A, relative steady-state growth rate of cultures of WT, tco89Δ, and tor1Δ cells in the presence and absence of rapamycin (200 ng/ml) was determined as for Fig. 2B. Error bars denote S.E.; *, p < 0.05 relative to treated WT. Inset recovery of WT, tco89Δ, and tor1Δ cells from rapamycin treatment was determined as for Fig. 1. B, cultures of WT and kog1Δ-pkog1^ts cells were grown for 2 days at either permissive (22 °C) or nonpermissive (37 °C) temperatures during which they were diluted to maintain logarithmic growth and subsequently treated or not with rapamycin (rap) (200 ng/ml). The relative steady-state growth rates were determined as for Fig. 2B. Error bars denote S.E.; *, p < 0.05. C, relative steady-state growth rates of WT, ego1Δ, gtr2Δ, tco89Δ, and kog1Δ-pkog1^ts cultures treated or not with rapamycin (200 ng/ml), at a starting A_{600 nm} of 0.05 for untreated or 0.1 for rapamycin-treated cultures, were determined as for Fig. 2B. Cultures were grown at 28 °C. Error bars denote S.E.; *, p < 0.05 relative to equivalent WT treatment. D, heterozygous KOG1/kog1Δ::KanMX diploids were sporulated, and the tetrads (10 and 9, respectively) were dissected onto solid medium comprising YPD or YPD supplemented with rapamycin (200 ng/ml). The resulting plates were incubated at 30 °C. The dissected spores were monitored by microscopy for 3 days, and their ability to proliferate was scored. The percentage and fraction (in parentheses) of total spores that failed to double, doubled three times or fewer, or formed colonies (too many cells to accurately estimate the number of doublings) on each medium are shown as a function of time after dissection. All tetrads resulted in only two visible colonies after 3 days, and these were all found to be wild type (i.e. sensitive to G418). We infer that the 50% of dissected spores that failed to form visible colonies were all kog1Δ::KanMX.