BAG6 deficiency induces mis-distribution of mitochondrial clusters under depolarization

Abstract

Accumulation of damaged mitochondria is implicated in a number of neurodegenerative disorders, including Parkinson's disease. Therefore, the machinery for mitochondrial quality control is important for the prevention of such diseases. It has been reported that Parkin- and p62/sequestosome 1 (SQSTM1)-mediated clustering and subsequent elimination of damaged mitochondria (termed mitophagy) are critical for maintaining the quality of mitochondria under stress induced by uncoupling agents such as carbonyl cyanide m-chlorophenyl hydrazone. However, the molecular mechanisms underlying mitochondrial translocation to the perinuclear region during mitophagy have not been adequately addressed to date. In this study, we found that BCL2-associated athanogene 6 (BAG6; also known as BAT3 or Scythe) is required for this process. Indeed, RNA interference-mediated depletion of endogenous BAG6 prevented Parkin-dependent relocalization of mitochondrial clusters to the perinuclear cytoplasmic region, whereas BAG6 knockdown did not affect the translocation of Parkin and p62/SQSTM1 to the depolarized mitochondria and subsequent aggregation. These results suggest that BAG6 is essential for cytoplasmic redistribution, but not for clustering, of damaged mitochondria.

Keywords: BAG6; CCCP; PINK1; Parkin; mitochondria; p62.

Figure 1

BAG6 knockdown disrupts translocation of mitochondria to the perinuclear region under depolarization. (A) Efficacy of BAG6 knockdown by siRNA in HeLa cells. HeLa cells were transfected with control or BAG6 siRNA. Three days after transfection, the expression of BAG6 protein was examined by western blotting. (B) Three days after transfection with siRNA, Flag‐Parkin‐transfected HeLa cells were treated with CCCP for 4 h and stained with anti‐TOMM20 (mitochondria; red), anti‐Flag (Parkin; green), and Hoechst (nucleus; blue). Scale bar represents 20 μm. (C) HeLa cells were transfected with Flag‐Parkin together with control or BAG6 siRNA #1, followed by treatment of 80 μg·mL⁻¹ antimycin A for 4 h. Cells were stained with anti‐TOMM20 (mitochondria; red), anti‐Flag (Parkin; green), and Hoechst (nucleus; blue). Scale bar represents 20 μm. (D) Immunocytochemistry with TOMM20 of BAG6 knockdown and control HeLa cells without ectopic expression of Parkin. Cells were treated with 20 μm CCCP for 4 h or DMSO (negative control). Scale bar represents 20 μm.

Figure 2

Effects of BAG6 siRNA on distribution and size of mitochondria in Flag‐Parkin‐transfected cells. (A) HeLa cells were transfected with Flag‐Parkin, together with control or BAG6 siRNA. The distance of each mitochondrion from the nucleus was measured. The numbers of mitochondria located < 1.5 and ≥ 1.5 μm from the edge of the nucleus were counted. We selected 15 control and 15 BAG6‐knockdown cells and measured 188 and 186 randomly selected mitochondria, respectively. (B) Data shown in (A) are presented as a bar graph. Chi‐squared test was used for statistical analysis. (C–F) HeLa cells were transfected with Flag‐Parkin and non‐targeting or BAG6 siRNA. Cells were treated with 20 μm CCCP for 4 h. Fluorescence intensities along the line shown in C are displayed in D. Magenta and blue lines indicate TOMM20 and Hoechst signals, respectively. d _max is the maximum distance between the nucleus and the furthest mitochondrial cluster in each cell. (E) Average d _max of knockdown and control cells [mean ± standard deviation (SD); n = 42]. Bars represent the mean ± SD (Student's t‐test). (F) Distributions of d _max of control cells (blue) and BAG6‐knockdown cells (orange) are presented in a bar graph. (G) Box‐and‐whisker plots showing the area of mitochondria from control and BAG6‐knockdown cells. n.s.: not significant.

Figure 3

BAG6 knockdown does not impair relocalization of p62/SQSTM1 to depolarized mitochondria. HeLa cells were transfected with siRNA and T7‐Parkin. After 4 h of CCCP treatment, the cells were immunostained for Flag‐p62/SQSTM1. Scale bar represents 20 μm.

Figure 4

BAG6 knockdown did not affect the mitochondrial clearance rate induced by CCCP. HA‐Parkin‐expressing cells were treated with or without BAG6 siRNA #1 and incubated with 20 μm of CCCP. Cells were harvested at the indicated time points and were subjected to SDS/PAGE followed by immunoblotting with antibodies for BAG6, actin, and TOMM20.

Figure 5

BAG6 was localized in the mitochondrial fraction. (A) Schematic diagram of the fractionation protocol. (B) Fractions were analyzed by western blotting. Tubulin, calnexin, and TOMM20 were used as markers for the cytoplasm, microsome, and mitochondria, respectively. The grouping of blots cropped from different parts of the same membrane. (C) Pure mitochondrial fractions isolated from HeLa cells were incubated with high‐salt buffer 1 m KCl, 600 mm mannitol, 20 mm HEPES‐KOH, pH 7.5, 2 mm MgCl₂, and 1 mm PMSF) for 15 min on ice and then centrifuged for 5 min at 10 000 g. The resultant high‐salt‐washed pure mitochondria were recovered as a pellet. The samples were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and immunoblotted for BAG6 and TOMM20, which was used as a control for mitochondria‐embedded protein.