The initiator caspase Dronc is subject of enhanced autophagy upon proteasome impairment in Drosophila

Abstract

A major function of ubiquitylation is to deliver target proteins to the proteasome for degradation. In the apoptotic pathway in Drosophila, the inhibitor of apoptosis protein 1 (Diap1) regulates the activity of the initiator caspase Dronc (death regulator Nedd2-like caspase; caspase-9 ortholog) by ubiquitylation, supposedly targeting Dronc for degradation by the proteasome. Using a genetic approach, we show that Dronc protein fails to accumulate in epithelial cells with impaired proteasome function suggesting that it is not degraded by the proteasome, contrary to the expectation. Similarly, decreased autophagy, an alternative catabolic pathway, does not result in increased Dronc protein levels. However, combined impairment of the proteasome and autophagy triggers accumulation of Dronc protein levels suggesting that autophagy compensates for the loss of the proteasome with respect to Dronc turnover. Consistently, we show that loss of the proteasome enhances endogenous autophagy in epithelial cells. We propose that enhanced autophagy degrades Dronc if proteasome function is impaired.

Figure 1

prosβ2 mutant cells accumulate ubiquitin-conjugated proteins. Shown are high magnification images (x100) of the posterior compartment of prosβ2 mosaic eye imaginal discs labeled for ubiquitin (a), FK2 (b) and FK1 (c). In this and all other figures, posterior is to the right. The FK2 and FK1 antibodies detect ubiquitin-conjugated proteins, but not free unconjugated ubiquitin. The left panels indicate the positions of the proteasome mutant cell clones by absence of GFP, the middle panels show the experiment (in magenta), and the right panels are the merged images of left and middle panels. White arrows mark a few cell clones as examples. Similar data were obtained for Mov34 mosaic discs (Supplementary Figure S1). Wild-type mosaic control discs do not show accumulation of ubiquitin (Supplementary Figure S2). Genotype: ey-FLP; prosβ2^EP3067 FRT80/ubi-GFP FRT80

Figure 2

Mutations in proteasome subunits result in cell death. Shown are high magnification images (x100) of the posterior compartment of prosβ2 mosaic eye imaginal discs labeled for cCsp-3 (a', a'') and FK2 (a''', a'''') to identify prosβ2 mutant cell clones. cCsp-3 labeling is increased in some, but not all, prosβ2 mutant cells (red in a' and a''). Genotype: ey-FLP; prosβ2^EP3067 FRT80/ubi-GFP FRT80

Figure 3

Diap1, but not Dronc, accumulate in proteasome mutant clones. Shown are high magnification images (x100) of the posterior compartment of prosβ2 (a and c) and Mov34 (b and d) mosaic eye imaginal discs labeled for Dronc (a and b) and Diap1 (c and d). FK2 labeling was used to identify mutant clones. The left panels indicate the positions of the proteasome mutant cell clones by absence of GFP. In the middle panels, the proteasome mutant cell clones are positively marked by FK2 labeling (in magenta). The right panels show the Dronc (a'' and b'') and Diap1 labelings (c'' and d'') in red. White arrows mark a few cell clones as examples. See also related Supplementary Figures S3 and S4. Genotype in (a and c): ey-FLP; prosβ2^EP3067 FRT80/P[ubi-GFP] FRT80. Genotype in (b and d): ey-FLP; FRT42D Mov34^k08003/FRT42D P[ubi-GFP]

Figure 4

Loss of Atg7 alone does not affect Dronc protein levels. Dronc labeling of Atg7 mosaic eye imaginal discs. Atg7 was either downregulated by RNAi (a) or genetically inactivated in mutant cell clones (b). Atg7-deficient clones are marked by absence of GFP (a', b'). White arrows highlight one clone in each panel as example. Dronc protein levels are unaltered in Atg7-deficient clones (a'', b''). Genotypes: (a) yw hsFLP; tub>GFP>Gal4/UAS-Atg7^RNAi (>denotes FRT). (b) yw eyFLP; FRT42D Atg7^d14/FRT42D P[ubi-GFP]

Figure 5

Dronc accumulates in clones simultaneously mutant for the proteasome and autophagy. Mosaic eye (a) and wing (b) imaginal discs doubly deficient for the proteasome and autophagy labeled for Dronc (red). Atg7 knockdown was induced by RNAi in prosβ2 mutant cell clones using the MARCM method. Mutant cell clones are positively labeled by GFP. Dronc accumulates in double-deficient cell clones. The cross was performed at 25 °C. Genotype: hs-FLP UAS-GFP tub-Gal4; UAS-Atg7^RNAi/+prosβ2^DTS7 FRT80B/tub-Gal80 FRT80B

Figure 6

Impaired proteasome function induces autophagic flux. Mov34 mosaic eye imaginal discs expressing GFP-mCherry-Atg8a as marker for autophagic flux. Mov34 mutant cells were identified by FK2 labeling (blue in a; gray in a'). White arrows mark representative Mov34 mutant cells as examples. Although there is little to no GFP labeling in Mov34 mutant cell clones (a''), all clones contain increased mCherry labeling (a''') suggesting that autophagic flux is enhanced in proteasome-deficient cells. Genotype: ey-FLP; FRT42D Mov34^k08003 /FRT42D ; patg8a>GFP-mCherry-Atg8a