Abstract
The Bcl-2 proapoptotic proteins Bax and Bak mediate the permeabilization of the mitochondrial outer membrane during apoptosis. Current models consider that Bax and Bak form pores at the mitochondrial outer membrane that are responsible for the release of cytochrome c and other larger mitochondrial apoptotic factors (i.e. Smac/DIABLO, AIF, and endoglycosidase G). However, the properties and nature of Bax/Bak apoptotic pores remain enigmatic. Here, we performed a detailed analysis of the membrane permeabilizing activity of Bax and Bak at the single vesicle level. We directly visualized that cBid-activated Bax and BakΔC21 can form membrane pores large enough to release not only cytochrome c, but also allophycocyanine, a protein of 104 kDa. Interestingly, the size of Bax and BakΔC21 pores is not constant, as typically observed in purely proteinaceous channels, but evolves with time and depends on protein concentration. We found that Bax and BakΔC21 formed long-lived pores, whose areas changed with the amount of Bax/BakΔC21 but not with cardiolipin concentration. Altogether, our results demonstrate that Bax and BakΔC21 follow similar mechanisms of membrane permeabilization characterized by the formation of protein-permeable pores of dynamic size, in agreement with the proteolipidic nature of these apoptotic pores.
Highlights
During apoptosis Bax/Bak release differently sized proteins out of the mitochondria
Current models consider that Bax and Bak form pores at the mitochondrial outer membrane that are responsible for the release of cytochrome c and other larger mitochondrial apoptotic factors (i.e. Smac/DIABLO, AIF, and endoglycosidase G)
Novel Minimalist Assay to Visualize and Analyze Protein Permeation Across Bax/Bak Pores—To attempt direct visualization of protein permeation across Bax and/or Bak pores and directly examine how the size of such putative pores formed by Bax and Bak may be regulated, we implemented a novel minimalist system. This system is based on four components: (i) functional recombinant Bcl-2 proteins [14, 18, 19, 39], (ii) giant unilamellar vesicle (GUV) containing a lipid mixture mimicking the mitochondrial outer membrane (MOM), (iii) proteinaceous and non-proteinaceous fluorescent size markers, and (iv) a time-lapse confocal fluorescence microscope
Summary
During apoptosis Bax/Bak release differently sized proteins out of the mitochondria. Results: The size of Bax/Bak pores depends on protein concentration. Current models consider that Bax and Bak form pores at the mitochondrial outer membrane that are responsible for the release of cytochrome c and other larger mitochondrial apoptotic factors (i.e. Smac/DIABLO, AIF, and endoglycosidase G). Our results demonstrate that Bax and Bak⌬C21 follow similar mechanisms of membrane permeabilization characterized by the formation of protein-permeable pores of dynamic size, in agreement with the proteolipidic nature of these apoptotic pores. CL concentration varies the propensity for Bax/Bak⌬C21 pore formation, without altering the actual pore size and the number of pores per GUV Based in these observations, we propose that during apoptosis the release of the complete set of mitochondrial apoptogenic proteins into the cytosol occurs via protein-permeable proteolipidic pores formed by Bax/Bak, which can be finely tuned by changes in the concentration of the active forms of Bax/Bak or the CL concentration
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