TOM20-mediated transfer of Bcl2 from ER to MAM and mitochondria upon induction of apoptosis

Abstract

In this work, we have explored the subcellular localization of Bcl2, a major antiapoptotic protein. In U251 glioma cells, we found that Bcl2 is localized mainly in the ER and is translocated to MAM and mitochondria upon induction of apoptosis; this mitochondrial transfer was not restricted to the demonstrator cell line, even if cell-specific modulations exist. We found that the Bcl2/mitochondria interaction is controlled by TOM20, a protein that belongs to the protein import machinery of the mitochondrial outer membrane. The expression of a small domain of interaction of TOM20 with Bcl2 potentiates its anti-apoptotic properties, which suggests that the Bcl2-TOM20 interaction is proapoptotic. The role of MAM and TOM20 in Bcl2 apoptotic mitochondrial localization and function has been confirmed in a yeast model in which the ER-mitochondria encounter structure (ERMES) complex (required for MAM stability in yeast) has been disrupted. Bcl2-TOM20 interaction is thus an additional player in the control of apoptosis.

Fig. 1. Bcl2 localization during the early phase of apoptosis.

A Confocal analysis of MFN1 (blue) and RTN3 (green) shows the close proximity of mitochondria and ER in U251 cells. B Electron microscopy in U251 cells enables the visualization of MAM, formed by the juxtaposition of ER and mitochondria (red arrowheads). C Cell fractionation is realized in U251 cells and fractions are analyzed by western blot (GM130: Golgi protein, RTN3: ER protein, TOM20: mitochondria protein) (H: homogenate, CM: crude mitochondria, M: pure mitochondria, C: cytosol). The blots are representative of three independent fractionation experiments. D U251 cells were treated by STS and total lysates are analyzed by western blot. E U251 cells treated by STS for 0, 2 and 4 h are fractionated as in (C). Fractions are analyzed by western blot. F U251 cells were treated by etoposide for 24 h and HeLa cells by STS for 4 h. Cell fractionation was realized as in (E) and Bcl2 localization was analyzed by western blot. The blots shown in D–F are representative of three independent experiments. G Bcl2–TOM20 and Bcl2–RTN3 interactions were observed by PLA in U251 treated by STS.

Fig. 2. Bcl2 forced targeting to ER does not alter its function nor interaction with TOM20.

A U251 were transfected by GFP-Bcl2, GFP-Bcl2cb5 (ER targeting), or GFP-Bcl2Maob (mitochondrial targeting) and mitochondria were stained by mitotracker Red CMXRos. B U251 cells were transfected as in (A) and cells were treated by STS for 4 h. DEVDase activity was measured in protein extracts, normalized by the protein concentration, and corrected by the activity measured in non-treated cells. C Stably transfected U251 cells were treated by STS and cell fractionation was performed as described in Fig. 1. Bcl2 localization was analyzed by western blot. The blots shown are representative from three independent experiments. D Transfected U251 cells were treated by STS and Bcl2–TOM20 interaction was measured by PLA.

Fig. 3. Bcl2–TOM20 interaction is inhibited by a competitive peptide.

A Epitope mapping suggests a major putative sequence of TOM20 interacting with Bcl2. The intensity of TOM20 interaction with Bcl2 is represented by the black diamonds and the gray triangles represent the intensity of TOM20 interaction with TOM22 along the TOM20 sequence. (OMM: outer mitochondrial membrane). B TBI-GFP was expressed in U251 cells then treated by STS (4 h). Bcl2–TOM20 interaction was measured by PLA. C U251 cells were transfected by a plasmid expressing the fluorescent proteins or peptides indicated. Cells were treated by STS and DEVDase activity was measured in the transfected cells by flow cytometry and expressed as the percentage of the activity measured in non-transfected cells. D GFP-tagged peptides were expressed in U251 cells later treated by the indicated drugs (see “Material and methods” for details). The protection index compared to the cell death induced in non-transfected cells was indicated on the graph (mean ± sd; *p < 0.05 and **p < 0.01 vs. non-transfected cells, ^#p < 0.05 and ^##p < 0.01 between TBI-GFP and BIRD2-GFP).

Fig. 4. Bcl2 interacts with TOM20 in yeast.

A Sequence homology between human and yeast TOM20 in the TBI sequence. B Yeast or human TOM20 expression wasinduced in S. cerevisieae together with human Bcl2. After 14 h, whole-cell extracts and isolated mitochondria were analyzed for Bcl-2 content by western blot. Mitochondrial porin (Por1) was used as a mitochondrial loading control. The ratio Bcl2/Por1 was quantified for each blot and the mitochondrial ratio was compared to the total ratio. C GFP-TBI expression was induced in yeast cells expressing human Bcl2, for 14 h, and isolated mitochondria and post-mitochondrial supernatants (PMS) were analyzed for Bcl-2 content. Mitochondrial porin (Por1) and Phosphoglycerate Kinase (Pgk1) were used as mitochondrial and cytosolic markers, respectively. D The expression of the constitutively active mutant BaxP168A was induced in wild-type yeast cells expressing human Bcl2 or the Δmdm34 mutant expressing human Bcl2. Mitochondria and PMS were analyzed for Bcl-2 content by western blotting.