Inactivation of Tap42 induces autophagy. (A) GFP-Atg8 processing is enhanced by inactivation of Tap42. Wild-type TAP42 (Y3033) and temperature-sensitive mutant tap42-109 (Y3035) cells expressing GFP-Atg8 were grown in SMD for 1 h at 23°C or 37°C and treated with or without rapamycin for 3 h. Proteins were precipitated with TCA, and resolved by SDS-PAGE followed by immunoblotting with anti-GFP antibody. (B) Deletion of ATG genes blocks processing of GFP-Atg8 induced by inactivation of Tap42. Protein extracts from the tap42-109 strain or the same strain harboring deletions in ATG1 (TYY205), ATG13 (TYY206) or ATG17 (TYY204) and expressing GFP-Atg8 were analyzed as in (A). (C) Tap42 inactivation stimulates Atg8 expression and conjugation. Protein extracts from TAP42 and tap42-109 cells grown at permissive or nonpermissive temperature were precipitated with TCA and separated by 12% SDS-PAGE in the presence of 6 M urea followed by immunoblotting with anti-Atg8 antiserum, or anti-Pgk1 antiserum as a loading control. (D) Selective autophagy is induced with inactivation of Tap42. Protein extracts from TAP42, tap42-11, tap42-106 and tap42-109 strains harboring deletions in VAC8 or both ATG1 and VAC8 and grown at permissive or nonpermissive temperature were analyzed by immunoblotting, using antiserum to Ape1.

Induction of autophagy by inactivation of Tap42 is allele-independent. (A) Protein extracts from tap42-11 (Y3032), tap42-106 (Y3034) or tap42-109 strains expressing GFP-Atg8 were analyzed as in Figure 1A. (B) The Pho8Δ60 assay was carried out in the wild-type (WT) and the indicated tap42 mutant strains at permissive and nonpermissive temperature. The error bars represent the standard deviation.

Autophagy induction by inactivation of Tap42 is dependent on Atg1 kinase activity. (A) Kinase-defective Atg1 mutants block autophagy when Tap42 is inactivated. Protein extracts from TAP42 and tap42-109 strains with a deletion of ATG1 and harboring either the empty vector, or a plasmid expressing wild-type Atg1, the Atg1^K54A, or Atg1^K54A,M102A mutant were grown at permissive or nonpermissive temperature in the absence or presence of rapamycin, and protein extracts were subjected to immunoblotting with anti-GFP antibody. (B) The tap42-109 atg1 Δ cells expressing plasmid-borne wild-type Atg1, or the Atg1^K54A or Atg1^K54A,M102A mutant were grown in SMD at permissive temperature to mid-log phase, shifted to nonpermissive temperature for 1.5 h and imaged by fluorescence microscopy. DIC, differential interference contrast.

Deletion of TIP41 suppresses the induction of autophagy that occurs with inactivation of Tap42. (A) Deletion of TIP41 suppresses the growth defect of tap42-109 cells. Serial dilutions of TAP42, tap42-109 and tap42-109 tip41 Δ (TYY224) cells were plated on YPD plates and incubated at 24°C, 30°C and 37°C for 2 days. (B) Deletion of TIP41 blocks the GFP-Atg8 processing that occurs with inactivation of Tap42. Protein extracts from TAP42, tap42-109 and tap42-109 tip41Δ cells expressing GFP-Atg8 were analyzed by immunoblotting, as described in Figure 1. (C) Deletion of TIP41 prevents the stimulation of Atg8 expression and conjugation that is seen with inactivation of Tap42. Protein extracts from TAP42, tap42-109 and tap42-109 tip41 Δ cells were analyzed by immunoblotting, as described in Figure 1.

Overexpression of Tip41 induces autophagy. (A) Overexpression of Tip41 inhibits cell growth. Wild-type (W303-1B) cells carrying a control vector (pTY006) or a plasmid containing TIP41 under control of the GAL1 promoter (pTY702) were streaked on glucose (SMD) or galactose (SMG) plates and incubated at 30°C for 3 days. (B) HA-tagged Tip41 is overexpressed from the GAL1 promoter. Wild-type cells carrying the control vector or pGAL1-TIP41 were grown in SMD and shifted to SMG. At the indicated times, TCA-precipitated proteins were subjected to immunoblotting with anti-HA antibody, as in Figure 1. (C) Overexpression of Tip41 stimulates GFP-Atg8 processing. Wild-type cells carrying the control vector or pGAL1-TIP41 were grown in SMD and shifted to SMG in the absence or presence of rapamycin. At the indicated times, TCA-precipitated proteins were subjected to immunoblotting with anti-GFP antibody. (D) Overexpression of Tip41 results in processing of GFP-Atg8 in an autophagy-dependent manner. Wild-type cells carrying the control vector or pGAL1-TIP41, and atg1Δ (TYY164) cells carrying pGAL1-TIP41 were grown in SMG for 8 h with or without rapamycin, and protein extracts were analyzed by immunoblotting.

Tap42-associated PP2A negatively regulates autophagy. (A) GFP-Atg8 processing is enhanced by inactivation of PP2A, whereas processing is normal in the absence of Sit4 activity. TAP42 (W303-1A), tap42-109 (Y3035), wild-type PPH22 (DEY213), temperature-sensitive mutant pph22-172 (DEY172-2B), wild-type SIT4 (TYY270), and temperature-sensitive mutant sit4-102 (TYY271) cells expressing GFP-Atg8 were grown in SMD for 1 h at 23°C or 37°C and treated with or without rapamycin for 3 h. TCA-precipitated proteins were analyzed by immunoblotting as in Figure 1. (B) Overexpression of PP2A inhibits GFP-Atg8 processing induced by rapamycin. Wild-type cells expressing GFP-Atg8 and carrying a control plasmid (pTY006, −) or a plasmid containing SIT4 (pTY703), PPH21 (pTY706) or PPH22 (pTY707) under control of the GAL1 promoter were grown in SMG for 8 h and treated with rapamycin. At the indicated times, TCA-precipitated proteins were subjected to immunoblotting as in Figure 1. Units represent the intensity of cleaved GFP. (C) Overexpression of PP2A containing a mutation in the catalytic site prevents inhibition of rapamycin-induced GFP-Atg8 processing. Wild-type cells expressing GFP-Atg8 and carrying a control plasmid (−), a plasmid encoding wild-type PPH22 (WT) or the Pph22^H186N mutant (H186N) and expressing GFP-Atg8 were grown in SMG for 8 h and treated with rapamycin. At the indicated times, TCA-precipitated proteins were subjected to immunoblotting as in Figure 1. Units represent the intensity of cleaved GFP. (D) Gln3 and Gat1 transcription factors are not needed for autophagy induction. Wild-type, atg1 Δ or gln3 Δ gat1Δ cells expressing GFP-Atg8 were grown in SMD to mid-log phase and treated with rapamycin as indicated. Protein extracts were analyzed by western blot as in Figure 1.

Conceptual model for Tap42-PP2A-regulated autophagy inhibition. The Tap42-PP2A pathway negatively regulates autophagy, and this process is antagonized by Tip41. Arrows indicate positive regulation and bars negative regulation. The target of PP2A is not known and the dashed bar indicates that Atg1 activity is needed for the autophagy induction that occurs when Tap42 or PP2A are inactivated, but not that Atg1 kinase is the direct target of this inhibition.