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. 2017 Mar 22;18(5):765–780. doi: 10.15252/embr.201643146

Figure 5. The pLIR2 sequence is new motif involved in the specific recognition of Atg8‐PE.

Figure 5

  1. The atg4Δ pep4Δ strain transformed with integration plasmids expressing Atg4 (SAY144), Atg4PD (SAY145), Atg4pLIR1 (SAY146), Atg4pLIR2 (SAY147), Atg4pLIR3 (RHY009), and Atg4pLIR4 (RHY010) or an empty plasmid (JSY163) was grown and starved as in Fig 1D before being processed for EM. Autophagic bodies (AB) are highlighted in the EM micrographs with asterisks. CW, cell wall; LD, lipid droplet; M, mitochondria; N, nucleus; PM, plasma membrane; V, vacuole. Scale bars, 1 μm.
  2. Quantification of the autophagic bodies. Average number of autophagic bodies (AB) per 50 vacuole sections ± SD. Significant differences (P < 0.05) between the Atg4 mutants and the WT were calculated using the paired two‐tailed Student's t‐test, and they are indicated with the symbol #.
  3. Quantification of the diameter of the AB. Average diameter of the AB in 50 vacuole sections ± SD. Significant differences (P < 0.05) between the various Atg4 mutants and the WT were calculated using the paired two‐tailed Student's t‐test, and they are indicated with the symbol #.
  4. The atg4Δ atg3Δ (FKY428) or the atg4Δ atg3Δ (FKY437) strains carrying the integrative GFP‐ATG8ΔR plasmid were transformed with the centromeric plasmids expressing either Atg4‐13xmyc, Atg4pLIR1‐13xmyc, Atg4pLIR2‐13xmyc, Atg4pLIR3‐13xmyc, or and Atg4pLIR4‐13xmyc. Strains were processed for pull‐down experiments as in Fig 2B. Quantification is shown in Fig 6A.
  5. Atg4, Atg4pLIR2, and Atg4pLIR4 were translated and radiolabeled in vitro as described in the Materials and Methods section before pull‐down (PD) with GST or GST‐Atg8 immobilized on glutathione‐beads. Beads were successively washed, and the eluted material was resolved by SDS–PAGE. Atg4 was visualized by autoradiographs, while GST and GST‐Atg8 amounts were assessed by Coomassie brilliant blue staining of the SDS–PAGE gel. The graph is the quantification of three independent experiments ± SD, where the binding of Atg4 is set as 100%.