Figure 4.

EDTP inhibits whereas Mtmr6 moderately promotes basal autophagy under nutrient-rich conditions. (A) Western blot analysis showing that soluble ref(2)P levels become lowered in fat body cells deficient in EDTP, but become highly elevated in cells defective for Mtmr6, as compared with control. ref(2)P serves as an autophagic substrate, thereby is widely used to monitor the autophagic degradation. (B) In Mtmr6 mutant genetic backgrounds, the amount of insoluble GFP-ref(2)P-containing protein aggregates becomes elevated. This change stems from the difference in the number of GFP-ref(2)P-positive structures and not from the alteration in the size of structures. Atg7^Δ77 mutant animals defective for autophagosome formation were also involved. (C) Clonal silencing of EDTP highly elevates the quantity of mCherry-Atg18a-positive early autophagic structures in Syx17^LL mutant fat body cells, which are deficient in autophagosome-lysosome fusion. Clonal green cells treated with RNAi are outlined by a white dotted line. Analysis was performed by using hsFLP; Syx17^LL, r4-mCherry-Atg18a, Act<CD2< Gal4, UAS-nlsGFP animals. (D) The ratio of hyperphosphorylated and non-hyperphosphorylated Atg13 levels is slightly decreased in EDTP mutant, but not altered in Mtmr6 mutant samples compared to the corresponding control, indicating that the activity of the induction complex is not enhanced by these genes. Atg8a-II/Atg8a-I ratio is not altered in fat body cells deficient in EDTP, but increased in Mtmr6 mutant samples. Atg8a-I is a cytosolic, Atg8a-II is a membrane-conjugated protein form. (E) Mutation of EDTP significantly elevates the quantity of GFP-2xFYVE-positive structures in Uvrag-RNAi cells, in which only the autophagy-specific PtdIns3K complex is active. GFP-2xFYVE bounds PtdIns3P and labels only early autophagic structures in Uvrag-downregulated cells. UAS-GFP-2xFYVE transgene is expressed by Cg-Gal4 driver. In panels A and D, αTub84B was used as an internal control. In panels A, B and D, “+” indicates w¹¹¹⁸ mutant control larvae. In panels B, C and E, Hoechst staining (blue) indicates nuclei, scale bar: 10 μm. Fluorescence microscopy images were composed of multiple optical sections. Quantifications are shown in box plots, *: p < 0.05, **: p < 0.01, ***: p < 0.001, ns: not significant. For statistics, see the Materials and Methods section. Fat body samples were prepared from well-fed animals at the third instar feeding larval (L3F) stage