Bax contains two functional mitochondrial targeting sequences and translocates to mitochondria in a conformational change- and homo-oligomerization-driven process

Abstract

The apoptosis gateway protein Bax normally exists in the cytosol as a globular shaped monomer composed of nine alpha-helices. During apoptosis, Bax translocates to the mitochondria, forms homo-oligomers, and subsequently induces mitochondrial damage. The mechanism of Bax mitochondrial translocation remains unclear. Among the nine alpha-helices of Bax, helices 4, 5, 6, and 9 are capable of targeting a heterologous protein to mitochondria. However, only helices 6 and 9 can independently direct the oligomerized Bax to the mitochondria. Although Bax mitochondrial translocation can still proceed with mutations in either helix 6 or helix 9, combined mutations completely abolished mitochondrial targeting in response to activating signals. Using a proline mutagenesis scanning analysis, we demonstrated that conformational changes were sufficient to cause Bax to move from the cytosol to the mitochondria. Moreover, we found that homo-oligomerization of Bax contributed to its mitochondrial translocation. These results suggest that Bax is targeted to the mitochondria through the exposure of one or both of the two functional mitochondrial targeting sequences in a conformational change-driven and homo-oligomerization-aided process.

FIGURE 1.

Individual helices of Bax serve as independent mitochondrial (Mito.) targeting sequences. Each of the nine Bax α-helices was cloned individually into the GFN2 vector as specified under “Experimental Procedure.” Individual Bax α-helix-GFP fusions were expressed in HeLa cells, and mitochondrial localization was monitored by co-localization with TOM20. The same phenotype was observed in all of the cells in the viewing field for any given construct.

FIGURE 2.

Targeting of the Bax homo-oligomerization domain to mitochondria by helix 6 and helix 9. A, homo-oligomerization of the Bax homo-oligomerization domain fused to different mitochondrial targeting sequences. A schematic representation of the Bax oligomerization domain is shown, with attachment of helix 6 and helix 9 on the left. Gel-filtration analysis of GFP (28 kDa), GFP-BaxH(2–5) (40 kDa), GFP-BaxH(2–5)-H9 (40 kDa), or H6-GFP-BaxH(2–5) (40 kDa) was carried out after transfection. B, mitochondrial localization of the homo-oligomerization domain fusion proteins. The same proteins used in A were expressed in HeLa cells and examined for their mitochondrial (Mito.) targeting in HeLa cells by co-localization with TOM20. The same phenotype was observed in all of the cells in the viewing field for any given construct.

FIGURE 3.

Requirement of helices 6 and 9 as functional Bax mitochondrial targeting sequences. GFP-Bax and G-Bax helix 6 and helix 9 mutants were transfected into HeLa cells in the absence or presence of tBid expression plasmid. Mitochondrial localization was determined by co-localization with TOM20. The same phenotype was observed in all of the cells in the viewing field for any given construct.

FIGURE 4.

Requirement of helices 6 and 9 for Bax pro-apoptotic activity of Bax. Bax^−/−Bak^−/− DKO MEFs were transfected with GFP, GFP-Bax, and G-Bax helix 6 and helix 9 mutants in the absence or presence of tBid. Apoptotic activity was determined by the percentage of GFP cells undergoing nuclear condensation as measured by staining with Hoechst 33342. The results are the mean percentage of apoptotic cells ± S.D. from at least three independent transfections.

FIGURE 5.

Conformational changes induce Bax mitochondrial translocation. A, the structure of Bax, taken from Suzuki et al. (25). B, schematic representation of the proline mutants of Bax. P represents the proline mutation. The amino acid residues for each proline mutant, specified under “Experimental Procedures,” were generated to occur at the midpoint of each helix. C, subcellular localization of Bax and its proline mutants. Each of the GFP-Bax proline mutants was transfected into Bax^−/−Bak^−/− cells, and mitochondrial localization was determined by co-staining with TOM20. The same phenotype was observed in all of the cells in the viewing field for any given construct. Mito., mitochondrial.

FIGURE 6.

Defective homo-oligomerization inhibits Bax mitochondrial translocation. A, gel-filtration analysis of Bax and Bax92–94A mutant. Expression plasmids expressing GFP, G-Bax, or G-Bax92–94A were transfected into HeLa cells with or without co-transfection of tBid expression plasmid. Whole-cell lysates were harvested and loaded onto a Superdex 200 column. Fractions were subjected to SDS-PAGE and analyzed by Western blot with GFP antibody. B, mitochondrial localization of Bax and Bax92–94A mutants in HeLa cells. Expression plasmids expressing GFP, G-Bax, or G-Bax92–94A were transfected into HeLa cells. These cells were either co-transfected with tBid expression plasmid or treated with UV light (20 J/m2). Four hours after UV treatment, these cells were immunostained with TOM20 antibody and visualized under a fluorescence microscope. The same phenotype was observed in all of the cells in the viewing field for any given construct.

FIGURE 7.

Model for the mitochondrial translocation of Bax. In this model, although conformational changes and mitochondrial translocation are reversible, homo-oligomerization is an irreversible process, which helps stabilize and maintain the active conformation of Bax and therefore drives the equilibrium toward mitochondrial translocation.