Mechanism and importance of mitochondrial import of BH3-only proteins during apoptosis
Final Report Abstract
BH3-only proteins are the main triggers of mitochondrial apoptosis: once activated or highly expressed, they activate the proteins Bax and/or Bak at mitochondria to release cytochrome c from the mitochondrial intermembrane space into the cytosol, where it initiates the activation of caspases, eventually leading to apoptosis. It appears a requirement of BH3-only protein function that they act directly at or in very close proximity of mitochondria but if and how they locate to mitochondria had not been systematically tested. In the work that formed the basis of this project we had observed that a minor isoform of one BH3-only protein (BimS) is inserted into the outer mitochondrial membrane (OMM) via its C-terminus. This project was designed to investigate the molecular mechanism and the potential importance of mitochondrial localisation and import of mammalian BH3-only proteins. In in vitro import assays, by microscopy and by analysis of intact cells expressing designed variants of BH3-only proteins we here showed that in five of seven tested BH3-only proteins (Bim, Puma, tBid, Bmf, Noxa) a C-terminal alpha-helix is necessary and sufficient to target the protein to and insert it into the OMM. One BH3-only protein (Bik) was targeted to the ER- membrane while one (Bad) appeared not to localise to intracellular membranes. Three BH3-only proteins (Bim, Puma, tBid) were able, when inserted into the OMM, to activate Bax. Closer analysis of Bim suggested that no other proteins were required. These results propose a model of mitochondrial apoptosis where ‘activator’ BH3-only proteins insert into the OMM, probably assume a certain tertiary structure and directly activate Bax, which presumably randomly diffuses to mitochondria. The contact with BH3-only proteins at the OMM allows Bax-activation and membrane insertion, leading to cytochrome c-release. This model will be tested in future studies. We further illustrated the importance of OMM-insertion for mouse Noxa protein: close proximity of the BH3-domain in Noxa to the OMM is conveyed by the C-terminal membrane anchor and is necessary for neutralisation and presumably binding to Mcl-1. We further found interaction of Bim with the components of the OMM import/translocase complex, Tom20 and Tom40. However, the two Tom-proteins appeared not to be required for Bim OMM-insertion and pro-apoptotic function. We believe that these results add to our understanding of mitochondrial apoptosis and provide a basis for future studies and perhaps manipulation of this process.
Publications
- (2012). BH3-only proteins are tail-anchored in the outer mitochondrial membrane and can initiate the activation of Bax. Cell Death Diff., 19(8):1328-36
Wilfling, F., Weber, A., Potthoff, S., Vögtle, F.N., Meisinger, C., Paschen, S. A. and Häcker, G.
(See online at https://doi.org/10.1038/cdd.2012.9)
