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

Figure 3. The Dsl1 complex is required for autophagy.

Figure 3

  1. Effect of ykt6 and dsl1 complex ts mutants on autophagosome–vacuole fusion. Cells carrying a CEN plasmid expressing GFP‐Atg8 were grown at 24°C in SGC and then shifted to SG‐N for 2 h at 24°C or 37°C. Vacuoles were stained by FM4‐64 and analyzed by fluorescence microscopy. Scale bar, 5 μm. Scale bar of inset is 0.6 μm.
  2. Analysis of autophagy over time. Cells were grown at 24°C and shifted to starvation medium at 24°C or 37°C for the indicated time periods. The cells were harvested, and proteins were extracted from cells by TCA precipitation. The extracted proteins were analyzed by SDS–PAGE and Western blotting against GFP.
  3. Protease protection assay of ykt6‐11 and dsl3 mutants. Lysates of atg1∆, ypt7∆, ykt6ts, and dsl3ts cells carrying a CEN plasmid expressing GFP‐Atg8 were grown at 37°C and subjected to the protease (PK) protection (see Materials and Methods for details).
  4. Localization of Atg8 relative to Atg1 during nitrogen starvation condition in sec12‐1 ts mutant. Cells carrying a CEN plasmid expressing GFP‐Atg8 and encoding 3xmCherry‐tagged Atg1 were grown at 24°C in SDC and then shifted to SD‐N for 2 h at 24°C or 37°C, and analyzed by fluorescence microscopy. Individual slices are shown. Scale bar, 5 μm. Scale bar of inset is 0.6 μm.
  5. Localization of Ykt6 relative to Dsl1 during growth and nitrogen starvation conditions. Cells expressing GFP‐tagged Ykt6 under the control of the PHO5 promoter and genomically 3xmCherry‐tagged Dsl1 were grown in SDC or SD‐N for 2 h and analyzed by fluorescence microscopy. Individual slices are shown. Scale bar, 5 μm.
  6. Percentage of Ykt6 puncta colocalizing with Dsl1. The data were quantified from (E). Error bars represent standard deviation of three independent experiments. Cells (n ≥ 250) and Dsl1 dots (n ≥ 50) were quantified.
  7. Localization of Dsl3 relative to Atg8 during growth and nitrogen starvation. Cells expressing GFP‐tagged Dsl3 and mCherry‐tagged Atg8 were grown in SDC or SD‐N for 2 h and analyzed by fluorescence microscopy. Scale bar, 5 μm.
Data information: Arrows in images indicate dot colocalization of Atg1 with Atg8 (D) and Ykt6 with Dsl1 (E).