The nitrogen starvation-induced inhibitor Rts3 restrains Sit4/PP6 to gate quiescence downstream of TORC1

Cellular quiescence is a reversible state essential for survival under nutrient-limiting or growth-restrictive conditions, yet the mechanisms fine-tuning its depth and reversibility remain elusive. Here, we identify Saccharomyces cerevisiae Rts3 as a regulator of the quiescence trajectory downstream of TORC1. Using phosphatase inhibitor beads and mass spectrometry, we characterize Rts3 as a phosphatase interactor in rapamycin-treated cells and define it as an inhibitor of the PP6 phosphatase Sit4. Mechanistically, it employs an α-helix to dock directly into the Sit4-Sap185/190 catalytic cleft. Transcriptionally induced by Gln3/Gat1 during nitrogen starvation, Rts3 is rapidly degraded upon nutrient repletion via a TORC1-SCF Cdc4 -proteasome axis. By selectively constraining Sit4-Sap185/190 activity, this inhibitor modulates nitrogen-responsive transcriptional and translational programs to prevent excessive accumulation of Gln3/Rtg3 targets, establishing a feedback loop gating quiescence depth to support long-term survival. Our findings position Rts3 as a dynamic molecular brake on Sit4, ensuring a protective yet reversible quiescent state.