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. 2020 Sep 10;9:e58073. doi: 10.7554/eLife.58073

Figure 2. Atg11(522-544) mediates a specific and direct interaction with the tMIT domain of Atg1.

(A) Coimmunoprecipitation assay showed that Atg11 physically interacts with Atg1. Atg11 and Atg1 were endogenously tagged with YFP and myc, respectively. (B) Pil1 co-tethering assay identified an interaction between Atg11(522-583) and the tMIT domain of Atg1. Log-phase cells co-expressing Pil1-CFP-Atg11(522-583) and an mCherry-tagged Atg1 fragment were examined by fluorescence microscopy. Scale bar, 3 μm. (C) Atg11(522-552) but not Atg11(532-583) interacted with the tMIT domain of Atg1 in the Pil1 co-tethering assay. Scale bar, 3 μm. (D) Y2H assay showed that Atg11(522-544) is sufficient for interacting with Atg1. Crb2, which can self-interact, served both as a positive control and a specificity control. (E) The sequence of S. pombe Atg11(522-544) was aligned to the corresponding regions of Atg11 proteins from three other fission yeast species. (F) Mutating either F526 or Y527 to alanine disrupted the autophagy function of Atg11(522-583). mYFP-Atg8 processing assay was performed as in Figure 1A. (G) In vitro GST pull-down assay using recombinant proteins demonstrated a direct interaction between Atg11(522-544) and the tMIT domain of Atg1.

Figure 2.

Figure 2—figure supplement 1. AP-MS analysis of Atg1-associated proteins and secondary structure prediction analysis on Atg11.

Figure 2—figure supplement 1.

(A) AP-MS analysis showed that Atg11, but not Atg13, Atg17, or Atg101, co-purified with Atg1. Endogenously YFP-tagged Atg1 was immunopurified, and purified proteins were identified using mass spectrometry. (B) The sequence of Atg11(511-550) was subjected to secondary structure prediction using the Quick2D tool in the MPI Bioinformatics Toolkit (https://toolkit.tuebingen.mpg.de; Zimmermann et al., 2018). (C) The sequence of Atg11(511-590) was subjected to coiled coil prediction using the MARCOIL tool in the MPI Bioinformatics Toolkit (Delorenzi and Speed, 2002; Zimmermann et al., 2018).