Figure 3.

The Atg9^F627A mutant blocks phagophore expansion. (A) The atg9∆ strain carrying Sec63-GFP and mCherry-Atg8, was transformed with both the pDP105 plasmid, which expresses giant Ape1 upon addition of cupper to the medium, and an empty vector (RGY760, atg9∆) or an integration plasmid expressing Atg9-13xMYC (RGY762, Atg9) or Atg9^F627A-13xMYC (RGY876, Atg9^F627A). The atg2∆ mutant carrying an integrative plasmid expressing Atg2[PM1]-TAP (RGY698, Atg2[PM1]) was used as control. For the giant Ape1 oligomers formation, cells were grown as described in the Materials and Methods section. Scale bars: 2 μm (main images) and 0.5 μm (insets). (B) The atg9∆ knockout in the W303 background, expressing mCherry-V5-Atg8 (RGY898, atg9∆), was transformed with an empty vector or integrative plasmids expressing GFP-tagged Atg9 (RGY900, Atg9) or Atg9^F627A (RGY901, Atg9^F627A), and imaged as in Figure 2C. The atg2∆ knockout in the W303 background expressing mCherry-V5-Atg8 and carrying an integrative plasmid expressing TAP-tagged version of Atg2[PM1] (RSGY042, Atg2[PM1]) was used as control. DIC, differential interference contrast. Scale bar, 2 μm. (C and D) Quantification of the average size in nm^2. (C) and intensity of the fluorescent signal in a.u. (D) of the mCherry-V5-Atg8 puncta in the experiment shown in panel B. Asterisks indicate significant differences with nitrogen-starved atg9∆ strain expressing the empty vector.