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-x(L) 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-x(L) 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.
Highlights
BH3-only proteins play a key role in the activation of Bax and Bak during apoptosis
Cells were cotransfected with two vectors, one encoding Mcl-1 or Bcl-xL fused to the N- or C-terminal fragment of Venus (VN-Mcl-1, VN-Bcl-xL, VC-Bcl-xL) and the other encoding Bim, PUMA, Bax, or Bak fused to the Nor C-terminal fragment of Venus (Fig. 1)
Deletion of the BH3 domain of Bim [16] or PUMA [17, 18] has been reported to abolish their proapoptotic effect, and we observed that ablation of this domain significantly reduced their binding to Mcl-1 (Fig. 2, A and B)
Summary
BH3-only proteins play a key role in the activation of Bax and Bak during apoptosis. Results: Interactions among Bcl-2 proteins have been detected in living cells. 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. Bcl-2 proteins are key players of apoptosis induction, regulating mitochondrial permeabilization and release of proteins that participate in the execution phase of apoptosis This family comprises anti- and proapoptotic members that share at least one of the four Bcl-2 homology (BH) domains BH1 to BH4 [1]. In this work we studied interactions among members of the Bcl-2 family during apoptosis in living cells using the bimolecular fluorescence complementation (BiFC) technique [9]. We demonstrate that Bim, PUMA, and Noxa can bind to Bax and Bak. Complexes of Bim with Bax are detected in cytosol and translocate to mitochondria prior to cell death. We found differences in the relative participation of the H1␣ and BH3 domains of Bax and Bak in the interaction with BH3only proteins
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
