Comparative regulation of autophagy through the TORC1, TORC2 and MAP kinase pathways. (A) Effect of TORC1, TORC2, PKA and MAP kinase pathway mutants on autophagy activation upon nitrogen depletion. MM cultures of strains CC10 (WT), CC31 (tsc1Δ tsc2Δ), CC18 (tco89Δ), CC21 (sck2Δ), CC66 (psk1Δ), CC16 (tor1Δ), CC33 (cgs1Δ), CC22 (pka1Δ), CC13 (sty1Δ) and CC23 (atf1Δ), 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 red circles. 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). (B) Effect of TORC1, TORC2 and MAP kinase pathway mutants on autophagy activation upon depletion of the glucose, sulfur or phosphorus sources. MM cultures of strains CC10 (WT), CC31 (tsc1Δ tsc2Δ), CC18 (tco89Δ), CC21 (sck2Δ) CC16 (tor1Δ), CC70 (gad8Δ) and CC13 (sty1Δ), all expressing GFP-Atg8, were shifted at the logarithmic phase (unt) to modified MM with low glucose (0.08% Gluc), or without sulfur (w/o S) or phosphorus (w/o P) sources for 8 h, and were processed and analyzed as in Figure 1A. A sample of extracts from CC10 (WT) after 2 h of nitrogen-starvation was used as an autophagy control. Numbers below the panels represent the amount of cleaved GFP relative to the amount in WT after 8 h under indicated conditions (0.08% Gluc, without [w/o] S and without [w/o] P) (with assigned values of 1). (C) Effect of TORC1, TORC2 and MAP kinase pathway mutants on bulk autophagy activation upon nitrogen depletion. MM cultures of strains RB44 (WT), RB40 (atg1Δ), RB45 (tsc1Δ), RB41 (tor2L1310P), RB48 (tco89Δ), RB39 (gad8Δ) and RB38 (sty1Δ), all expressing Pgk1-GFP, were shifted or not at the logarithmic phase (unt) to nitrogen-free medium for 1–2 h, and were processed and analyzed as in Figure 1A. Numbers below the panels represent the amount of cleaved GFP relative to the amount in WT after 1 h under nitrogen deprivation (with an assigned value of 1). (D) Effect of TORC1, TORC2 and MAP kinase pathway mutants on bulk autophagy activation upon depletion of the glucose, sulfur or phosphorus sources. MM cultures of strains strains RB44 (WT), RB40 (atg1Δ), RB45 (tsc1Δ), RB41 (tor2L1310P), RB48 (tco89Δ), RB39 (gad8Δ) and RB38 (sty1Δ), all expressing Pgk1-GFP, were shifted at the logarithmic phase (unt) to modified MM with low glucose (0.08% Gluc), or without sulfur (w/o S) or phosphorus (w/o P) sources for 8 h, and were processed and analyzed as in Figure 1A. Numbers below the panels represent the amount of cleaved GFP relative to the amount in WT after 8 h under each condition (0.08% Gluc, without [w/o] S and without [w/o] P) (with assigned values of 1). (E) Scheme depicting the impact of mutations at the TORC1, TORC2 or MAP kinase pathways on autophagy induction. Accelerated nutrient starvation-dependent autophagy of some mutants is indicated in green, while deficient autophagy is indicated in red. See text for details.