Fig. 5.

Import of the DJ-1 mutants requires mitochondrial protein translocases TOMM40 and TIMM23. (A) siRNA-mediated reduction in expression of endogenous TIMM23 and TOMM40. DJ-1 KO HeLa cells expressing Su9–GFP were treated with control, TIMM23, or TOMM40 siRNAs, and then immunoblotted using the indicated antibodies. To assess the knockdown efficiency, 10% of the control siRNA treated cell lysate was also loaded. The precursor and mature forms of Su9–GFP are shown as p and m. Immunoblots are representative of two independent experiments. (B,C) DJ-1 KO HeLa cells pre-treated with control, TIMM23 or TOMM40 siRNA were transfected with Su9–GFP (B) or WT DJ-1 (C), and then subjected to immunocytochemistry. (D,E) For quantitative analysis of the colocalization of GFP or DJ-1 with TOMM20, the Pearson correlation coefficient in individual cells was calculated, with quantitative analyses done using data in B and C. (F–I) DJ-1 KO HeLa cells pre-treated with control, TIMM23, or TOMM40 siRNA were transfected with E18K (F,H) or E18H (G,I) DJ-1 mutants and then subjected to immunocytochemical and quantitative analyses as in B–E. *P<0.01, N.S., not significant compared to control (one-way ANOVA with Sidak's correction). Box plots are as described in the Materials and Methods section. Representative images of two independent experiments are shown in B, C, F and G. Scale bars: 10 µm.