Figure 2. Sea3-GATOR1 attenuates TORC1 signaling via the Gtr1–Gtr2 Rag GTPases.

(A–E) Wild-type and the indicated mutant strains were grown in liquid EMM medium and their serial dilutions were spotted onto the plates. (A) Sea3 is required for normal cell growth. The indicated strains were spotted onto solid YES medium with (Rapamycin) or without (–) 100 ng/ml rapamycin. (B) Loss of Any1 rescues the growth defect of the sea3∆ and iml1∆ mutants. The indicated strains were spotted onto solid YES medium. (C) Sea3 and the GATOR1 complex function in the same pathway. Growth of the indicated strains was compared on solid EMM medium. (D) Sea3 attenuates TORC1 in parallel with the TSC complex. The indicated strains were spotted onto solid YES medium with (Rapamycin) or without (–) 100 ng/ml rapamycin. The growth defect of cells lacking Sea3 was accentuated in the absence of the TSC complex. (E) Genetic interactions between Sea3/Iml1 and Gtr1–Gtr2. The indicated strains were spotted onto solid YES medium. The expression of GDP-bound Gtr1 (gtr1SN) rescues the growth defect of the sea3∆ mutant. (F, G) Inactivation of TORC1 upon nitrogen starvation is delayed in cells lacking Sea3. TORC1 activity was monitored by detecting the TORC1-dependent phosphorylation of Psk1 (P-Psk1). The indicated strains were grown in EMM at 30°C, followed by shifting to the same medium without nitrogen source (–N). Cells were collected at the indicated time points after shifting to nitrogen starvation medium for immunoblotting. The samples were also probed with the anti-Psk1 antibody (Psk1), as well as the anti-Spc1 MAPK antibody for a loading control (LC).