Figure 6.
Drosophila ref(2)P is required for parkin-mediated suppression of the phenotype caused by protein conformational stress in mitochondria. (a) Immunoprecipitation of Parkin/p62 complexes in cultured cells. Human HEK-293 cells were transfected with the indicated constructs and incubated for 16 h. Lysates were subjected to immunoprecipitation using an anti-FLAG antibody and analysed by western blotting as indicated. (b) ref(2)POD2 and ref(2)P OD3 mutants exhibit mitochondrial defects in the indirect flight muscles. Ultrastructural defects in the thoracic muscles were analysed by TEM. Arrowheads point to mitochondrial vacuoles in the mutants. (c) ref(2)P was required for the Parkin-mediated suppression of the motor impairment in dOTC-expressing flies. The 3-day-old flies with the indicated genotypes were tested using a standard climbing assay (mean±S.D., n≥60 flies for each genotype). Statistically significant values relative to control are indicated (one-way ANOVA with Dunnett's multiple comparison test). (d) ref(2)P is required for Parkin-mediated suppression of mitochondrial protein loss in dOTC-expressing flies. The mitochondrial protein loss was addressed by measuring the levels of the nuclear-encoded subunit of mitochondrial complex I (NDUFS3). Flies were analysed by western blotting using the indicated antibodies