Pexophagy is blocked in cells lacking Slt2p but not the other MAPKs. (a) Peroxisomes were induced by growing wild-type (WT), atg1Δ, and slt2Δ strains expressing Pot1p-GFP in oleate medium to mid–log phase. Subsequently, cells were transferred to SD-N medium to trigger peroxisome degradation (pexophagy) for 24 h. Samples were taken at the indicated time points, and GFP cleavage was analyzed by immunoblotting. (b) Wild-type, atg1Δ, and slt2Δ strains expressing Pot1p-GFP were grown in oleate in the presence of the vacuolar stain, FM4-64, to mid–log phase and incubated in SD-N medium (pexophagy) for 6 h. The differential interference contrast (DIC) and GFP images are shown. Bar, 2 µm. (c) More than 100 cells per strain were examined by fluorescence microscopy for the number of peroxisomes in oleate and SD-N medium (pexophagy) for 6 h. (d) Wild-type, slt2Δ, atg5Δ, and atg24 strains were grown in oleate medium and induced for pexophagy in SD-N medium for 2 h. Postnuclear membrane fractions were either untreated (−), treated with proteases (proteinase K and trypsin; +), or proteases and detergent (Triton X-100; + +). Samples were processed and immunoblotted for Inp2p and Pot1p. (e) All six known MAPK deletion strains along with wild type were tested for pexophagy as described in a. All experiments were repeated at least thrice.

PKC1 pathway mutants are defective in pexophagy but not in autophagy and Cvt pathways. (a) Diploid wild-type (PKC1/PKC1), PKC1/pkc1Δ (heterozygote), wild-type (WT; haploid), atg1Δ, bck1Δ, and mkk1Δ mkk2Δ strains were analyzed for pexophagy. Cells were grown to mid–log phase in oleate medium, transferred to SD-N medium (pexophagy), and samples were collected at the indicated time intervals and analyzed for the peroxisomal marker, Pot1p, by immunoblotting. Numbers refer to the percentage of Pot1p at the various times. (b) Ape1p maturation assay was performed for diploid wild-type (PKC1/PKC1), PKC1/pkc1Δ (heterozygote), wild-type (haploid), atg1Δ, bck1Δ, and mkk1Δ mkk2Δ strains as described in Fig. 2 a. (c) For the GFP-Atg8p–processing assay, diploid wild-type (PKC1/PKC1), PKC1/pkc1Δ (heterozygote), atg1Δ, bck1Δ, and mkk1Δ mkk2Δ cells were treated as in Fig. 2 b and subjected to immunoblotting. All experiments were repeated thrice.

Cells lacking the cell surface sensor Mid2p are defective in pexophagy and a schematic representation of MAPK components involved in pexophagy. (a) Wild-type (WT), wsc1Δ, wsc2Δ, wsc3Δ, wsc4Δ, mtl1Δ, and mid2Δ cells were analyzed for pexophagy as described in Fig. 3 a. (b) MID2, PKC1, BCK1, MKK1 + MKK2, and SLT2 are all necessary for pexophagy. Our work suggests that Mid2p may be involved in sensing pexophagy signals that are eventually relayed to the classical SLT2 MAPK cascade consisting of upstream kinase MAPKKKK, Pkc1p that activates MAPKKK Bck1p. Bck1p then activates MAPKKs Mkk1p and Mkk2p, which phosphorylate the MAPK Slt2p on its T190 and Y192 residues. Subsequently, the kinase activity of phosphorylated Slt2p is required for pexophagy.

Autophagy and Cvt pathways are unaffected in the slt2Δ strain. (a) Wild-type (WT), atg1Δ, and slt2Δ strains were grown in SD medium to mid–log phase and placed in SD-N medium (starvation). At the indicated time intervals, samples were taken and analyzed for Ape1p maturation by immunoblotting. (b) Assay for autophagy monitoring GFP-Atg8p processing in wild-type, atg1Δ, and slt2Δ strains. Cells were treated as in a and were subjected to immunoblotting. All experiments were repeated thrice.

Kinase-dead and phosphorylation mutants of Slt2p are affected in pexophagy. Peroxisomes in wild-type (WT) and mutant cells were induced by incubating cells in oleate medium overnight, and pexophagy was triggered by transferring them to SD-N medium (pexophagy). Immunoblotting for the peroxisomal marker Pot1p was performed as described in Fig. 3 a. Phosphorylation of Slt2p and total Slt2p were monitored by immunoblotting using phospho-Slt2p and total Slt2p antibodies. The slt2Δ cells complemented with either the SLT2 ORF (slt2Δ + SLT2), the K45R mutation (slt2Δ + SLT2 K54R), or the T190A and Y192A mutations (slt2Δ + SLT2 TAYF) are shown. All experiments were repeated thrice.