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. 2020 Oct 7;21(12):e50733. doi: 10.15252/embr.202050733

Figure 7. The Atg1 kinase complex controls the fusogenic activity of Ykt6 during autophagy.

Figure 7

  1. Growth test of Ykt6 wild type and mutants. ykt6∆ cells carrying a URA3 plasmid coding for wild‐type Ykt6 and pRS413‐GAL1pr‐YKT6-GFP (wild type or mutant) were spotted as serial dilutions on SGC‐HIS to select for the mutant plasmid or 5‐fluoroorotic acid (5‐FOA) to force the loss of the plasmid coding for wild‐type Ykt6.
  2. Effect of Ykt6 phosphomutants on autophagy. ykt6∆ cells expressing wild‐type Ykt6 plus a pRS413 plasmid encoding GAL1pr‐YKT6 (wild type or mutants with S to A mutations [SA; S182, S183A] or S to D mutations [SD; S182D, S183D]) and a plasmid expressing GFP‐Atg8 were grown in SGC and then shifted to SG‐N for 2 h. Cells were analyzed by fluorescence microscopy. Individual slices are shown. Scale bar, 5 μm.
  3. Quantification of Atg8 dots per cell from images in (B). Error bars represent standard deviation of three independent experiments. Cells (n ≥ 100) and Atg8 dots (n ≥ 50) were quantified.
  4. Effect of Atg1‐Atg13 subcomplex on autophagosome–vacuole fusion in vitro. The vam3∆ strain carrying pRS413 plasmid encoding GAL1pr‐YKT6 (wild type or mutant), and expressing Atg9‐3xFLAG and GFP‐Atg8, was starved for 3 h at 30°C before purifying autophagosomes. Vacuoles were isolated from pep4∆ cells expressing Vac8‐3xmCherry and then incubated with autophagosomes in the presence or absence of 50 nM Atg1‐Atg13 subcomplex at 26°C for 1 h with or without ATP (Gao et al, 2018b). Scale bar, 5 μm.
  5. Quantification of (D). Error bars represent standard deviation of 3 independent experiments. Fluorescence intensity of GFP‐Atg8 in the vacuolar lumen was quantified by ImageJ using the ROI (region of interest) manager tool. Vacuoles (n ≥ 40) for each experiment were quantified.
  6. Working model for the recruitment and regulation of Ykt6 during autophagy. Ykt6 arrives either after prenylation of via COPI‐coated vesicles at the ER, where it binds the Dsl1 complex. Ykt6 is packaged into COPII‐coated vesicles, which deliver Ykt6 independently from Atg9 vesicles to the PAS. Atg1–Atg13 kinase controls Ykt6 activity until autophagosomes are completely closed.