Direct Interaction of Bax and Bak Proteins with Bcl-2 Homology Domain 3 (BH3)-only Proteins in Living Cells Revealed by Fluorescence Complementation |
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Authors: | Laura Vela Oscar Gonzalo Javier Naval Isabel Marzo |
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Institution: | From the Department of Biochemistry, Molecular and Cell Biology, Facultad de Ciencias, Universidad de Zaragoza, 5009 Zaragoza, Spain |
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Abstract: | The key event in the mitochondrial pathway of apoptosis is the activation of Bax and Bak by BH3-only proteins through a molecular mechanism that is still a matter of debate. Here we studied interactions among anti- and proapoptotic proteins of the Bcl-2 family in living cells by using bimolecular fluorescence complementation analysis. Our results indicate that the antiapoptotic proteins Mcl-1 and Bcl-xL bind preferably to the BH3-only proteins Bim, PUMA, and Noxa but can also bind to Bak and Bax. We also found a direct interaction between Bim, PUMA, or Noxa with either Bax or Bak during apoptosis induction. In HeLa cells, interaction of Bim with Bax occurs in cytosol, and then Bim-Bax complexes translocate to mitochondria. Complexes of either PUMA or Noxa with Bax or Bak were always detected at mitochondria. Overexpression of Bcl-xL or Mcl-1 delayed Bim/Bax translocation to mitochondria. These results reveal the ability of main BH3-only proteins to directly activate Bax and Bak in living cells and suggest that a complex network of interactions regulate the function of Bcl-2 family members during apoptosis. |
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Keywords: | Apoptosis Bax Bcl-2 Family Cell Death Mitochondrial Apoptosis BH3-only Bax/Bak Activation Bimolecular Fluorescence Complementation (BiFC) |
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