Fig. 5.
The PKA and Tor pathways independently target Atg13 to control autophagy activity. (A) Simultaneous inactivation of the PKA and Tor pathways resulted in an elevated autophagy response relative to the loss of either pathway alone. Autophagy levels were assessed with the ALP-based assay in tpk1-as cells that had been treated for the indicated time with either 25 μM 1NM-PP1, 200 ng/mL rapamycin, or both reagents. The data shown are from a single experiment that was representative of at least three independent replicates. (B) The PKA phosphorylation of Atg13 was not diminished upon inactivation of the Tor pathway. Atg13 was immunoprecipitated from cells following a 2 h treatment with either 20 (Lo) or 200 (Hi) ng/mL rapamycin. The level of PKA phosphorylation was subsequently assessed by Western blotting with the α-PKA substrate antibody. (C) Inactivation of the Ras/PKA pathway did not influence the Tor-dependent phosphorylation of Atg13. Cell extracts were prepared from the indicated cells and the level of Tor-dependent phosphorylation of Atg13 was assessed by Western blotting. In the left-hand lanes, cells containing either MET3-RAS2ala22 (A22) or a control plasmid (-) were incubated in methionine-free medium for 6 h to allow for expression from the MET3 promoter. In the middle lanes, the tpk1-as strain, PHY4710, was incubated for 4 h with 0 or 5 μM 1NM-PP1. The bottom panel in the middle lanes shows the level of PKA phosphorylation on Atg13 as assessed by Western blotting with the α-PKA substrate antibody. Note that the relative spread of the Atg13 “smear” is dependent upon the running conditions of the gel. (D) A model depicting the proposed roles of the PKA and Tor pathways in the control of autophagy. The dashed lines indicate the Atg1 and Atg13 interactions with each other or the PAS (and perhaps Atg17), and the solid lines indicate the regulatory effects of the PKA and/or Tor pathways on these interactions. See the text for additional details.