Figure 5.

The MAP kinase Sty1 does not regulate TORC1-dependent growth promotion functions, nor affects phosphorylation levels of Atg1 and Atg2, but positively regulates autophagy. (A) Sty1 is phosphorylated upon nitrogen or glucose starvation. MM cultures of wild-type strain CC10 were grown and shifted or not from the logarithmic phase (-) to modified MM without nitrogen (w/o N) or with low glucose (0.08%). As a control, logarithmic cultures were treated 10 min with 1 mM H₂O₂. TCA extracts were analyzed by western blot using anti-phospho-MAPK/p38 (Sty1-P) and anti-Sty1 antibodies. (B) Lack of Sty1 does not impair dephosphorylation of Psk1 and Rsp6 after nitrogen depletion. MM cultures of strains CC10 (WT) and CC13 (sty1Δ) were shifted or not to nitrogen depleted media for 10–30 min and analyzed as in Figure 2A. (C) Synergistic role of TORC1 and MAP kinase pathways on autophagy activation. MM cultures of strains CC10 (WT), CC14 (tsc1Δ), CC13 (sty1Δ) and CC111 (tsc1Δ sty1Δ), all expressing GFP-Atg8, were shifted or not to nitrogen-free medium for 2–7 h, and processed and analyzed as in Figure 2 C. 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) Lack of Sty1 does not impair dephosphorylation of Atg1 after nitrogen depletion. MM cultures of strains CC99 (WT) and CC110 (sty1Δ), both expressing Atg1-HA, were shifted or not to nitrogen depleted media for 1–2 h and analyzed as in Figure 2A, using anti-HA antibody. Numbers below the panels represent the amount of Atg1 relative to the amount in WT under physiological conditions (-) (with an assigned value of 1). (E) Lack of Sty1 does not impair phosphorylation of Atg2 after nitrogen depletion. MM cultures of strains CC122 (WT) and CC123 (sty1Δ), both expressing Atg2-HA, were shifted or not to nitrogen depleted media for 1–2 h and analyzed as in Figure 2A, using anti-HA antibody.