Figure 3. Shp1 affects autophagosome biogenesis. (a and b) Fluorescence microscopy of starved cells showed defective vacuolar uptake of GFP-Atg8. No GFP-Atg8–positive autophagosomes accumulated in the cytosol. (c) Electron microscopy of starved shp1Δ cells showed no vacuolar accumulation of autophagic bodies. (d) Lysed spheroplasts of starved cells were trypsin digested with and without detergent. Immunoblots with GFP antibodies showed proteolysis-resistant GFP-Atg8 (inside autophagosomes) in ypt7Δ but not in wild-type and shp1Δ cells. GFP-Atg8 breakdown yields GFP*. (e) Cells with a GFP-Atg8–positive PAS punctum were scored in fluorescence microscopy. The mean and SD of two experiments are shown, with >200 cells analyzed per strain. (f) To analyze Atg8-PE, extracts were separated in 6 M urea SDS-PAGE and immunoblotted with anti-Atg8.
Figure 3.

Shp1 affects autophagosome biogenesis. (a and b) Fluorescence microscopy of starved cells showed defective vacuolar uptake of GFP-Atg8. No GFP-Atg8–positive autophagosomes accumulated in the cytosol. (c) Electron microscopy of starved shp1Δ cells showed no vacuolar accumulation of autophagic bodies. (d) Lysed spheroplasts of starved cells were trypsin digested with and without detergent. Immunoblots with GFP antibodies showed proteolysis-resistant GFP-Atg8 (inside autophagosomes) in ypt7Δ but not in wild-type and shp1Δ cells. GFP-Atg8 breakdown yields GFP*. (e) Cells with a GFP-Atg8–positive PAS punctum were scored in fluorescence microscopy. The mean and SD of two experiments are shown, with >200 cells analyzed per strain. (f) To analyze Atg8-PE, extracts were separated in 6 M urea SDS-PAGE and immunoblotted with anti-Atg8.

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