Proteasome impairment leads to accumulation of cytoplasmic aggregates and enhances autophagic flux. A) Overexpressed GFP-Atg8a reporter is incorporated into the large protein aggregates containing p62 in Rpn2 RNAi cells. B) The tandemly tagged mCherry-GFP-Atg8a reporter forms numerous small mCherry-labeled autolysosomes, in addition to large aggregates positive for both mCherry and GFP in Rpn2 RNAi cells. C) The lysosome inhibitor chloroquine blocks autophagy-dependent quenching of GFP, as now most puncta are positive for both mCherry and GFP. D) Quantification of data from panels B and C (u or t test, n = 5-8 per genotype, ** P < 0.01, * P < 0.05), and error bars denote standard error. E) Transmission electron microscopy reveals the presence of autolysosomes (arrow) and large protein aggregates in fat body cells with impaired proteasome function in well-fed larvae. The boxed area is shown enlarged in panel E’. F) Quantification of ultrastructural data. Protein aggregates occupy 7% of the total cytoplasm in Rpn2 depleted fat body cells of well-fed larvae, and double-membrane autophagosomes (AP) and degrading autolysosomes (AL) take up 0.06% and 0.24% of the total cytoplasm, respectively. No such structures are recognized in fat body cells of well-fed control larvae (u test, n = 3 per genotype, ** P < 0.01), and error bars denote standard error. Boxed areas in A-C are shown enlarged. Scale bar in A equals 20 μm for A-C. Scale bars equal 1 μm in E, E’.