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. 2009 Jun 12;5(6):e1000515. doi: 10.1371/journal.pgen.1000515

Figure 1. A genetic screen for regulators of TORC1.

Figure 1

(A) A schematic of the TORC1 pathway. In nutrient rich environment, TORC1 is active and Gln3/Gat1 are in the cytoplasm. Upon TORC1 inactivation by rapamycin or amino acid starvation, Gln3 and Gat1 translocate into the nucleus, leading to the transcriptional activation of Dal80. (B) Dal80 gene expression reporter contains 600 basepairs of Dal80 promoter driving the expression of GFP. HygB, hygromycin B resistance gene, is used for selection of transformants. (C) Accumulation of GFP in cells is linear. Wildtype cells transformed with Dal80pr-GFP plasmid were treated either with rapamycin (20 ng/mL) or inoculated into nitrogen free medium. At indicated timepoints the average GFP content of the cells was determined by flow cytometry. (D) Fifty-five 96-well plates, containing all non-essential yeast deletion strains, were transformed with the Dal80pr-GFP reporter plasmid. The cells in each plate were grown under 3 conditions: YPD for 6 hours, YPD+rapamycin for 15 hours or N-free media for 4 hours. GFP content was determined by flow cytometry.