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. 2021 Jun 23;18(2):375–390. doi: 10.1080/15548627.2021.1935522

Figure 2.

Figure 2.

TORC1 negatively regulates autophagy. (A) TORC1 kinase activity and phosphorylation of its downstream consecutive targets, Psk1 and Rps6, are inhibited upon nutrient deprivation. MM cultures of strain CC10 (expressing GFP-Atg8) were collected at the logarithmic phase (-) and 30 min or 8 h after switching to modified MM with low glucose (0.08% Gluc), or without nitrogen (- N), sulfur (- S), and phosphorus (- P) sources. TCA extracts were analyzed by western blot with anti-phospho-Psk1 (anti-RPS6KB/p70) and anti-phospho-Rps6 (anti-AKT) antibodies. (B) Effect of TORC1 pathway mutants on the inhibition of Psk1 and Rsp6 phosphorylation after nitrogen depletion. MM cultures of strains CC10 (WT), JUP1350 (tor2L1310P), CC31 (tsc1Δ tsc2Δ), CC18 (tco89Δ) and CC21 (sck2Δ) were shifted to nitrogen depleted media for 10–30 min and analyzed as in (A). (C) Effect of TORC1 pathway mutants on autophagy activation upon nitrogen depletion. MM cultures of strains CC10 (WT), CC14 (tsc1Δ), CC31 (tsc1Δ tsc2Δ), CC18 (tco89Δ), CC66 (psk1Δ), CC89 (sck1Δ) and CC21 (sck2Δ), all expressing GFP-Atg8, were shifted or not at the logarithmic phase (unt) to nitrogen-free medium for 1–3 h, and were processed and analyzed as in Figure 1A. Accelerated GFP-Atg8 cleavage is indicated with green circles, while deficient cleavage is labeled with a red circle. Numbers below the panels represent the amount of cleaved GFP relative to the amount after 2 h without nitrogen in WT (with an assigned value of 1). (D) Atg1 dephosphorylation after nitrogen depletion is defective in tor2L1310P and tsc1Δ mutants. MM cultures of strains CC99 (WT), CC115 (tor2L1310P), CC101 (tsc1Δ), RB15 (tco89Δ), RB5 (psk1Δ), RB13 (sck1Δ), and RB6 (sck2Δ), all expressing Atg1-HA, were shifted or not to nitrogen-free medium, and processed and analyzed as in (A), using anti-HA antibody. (E) Scheme depicting the nutrient depletion-dependent antagonistic regulation by TORC1 pathway of growth-related functions and of autophagy. Deletion or point mutants used in this study are represented in red (low autophagy) and green (high autophagy). See text for details.