Investigation of the role of the C-terminus of Bax and of tc-Bid on Bax interaction with yeast mitochondria.

Because of structural homology with the transmembrane domain of Bcl-2, the proapoptotic protein Bax has been proposed to be anchored to the outer membrane of mitochondria through its carboxy-terminal end (CT). We took advantage of the absence of Bcl-2 family members in yeast to further investigate the role of Bax CT in its mitochondrial association and function. The complete deletion or the addition of a C-terminal c-myc tag as well as the replacement of CT by a random coiled sequence enhanced membrane insertion of Bax. It has previously been suggested that conformational change in the N-terminal end of Bax would allow the C-terminal end to play its anchoring function. We found that a mutant truncated in both N- and C-termini still exhibited a strong binding activity to mitochondria. In mammals, Bax interaction with the caspase-8-generated truncated form of Bid (tc-Bid) is believed to promote a conformational change necessary for the insertion of Bax into mitochondria. We coexpressed Bax and tc-Bid in yeast and found that native Bax functions are not stimulated by tc-Bid, whereas functions of an active variant with a modified CT are. We propose that Bax CT has to undergo a conformational change to allow the insertion of Bax in mitochondria but, contrary to current views, is not a bona fide membrane anchor.