Investigation of bar-induced release of cytochrome c from yeast mitochondria - Permeability of mitochondrial membranes, role of VDAC and ATP requirement

Recent studies that attempt to explore the action of pro- and anti-apoptoti c proteins of the bcl2 family demonstrate the crucial role of relocalizatio n of cytochrome c from the mitochondrial intermembrane space to the cytosol . This early event of apoptosis can be mimicked in the yeast Saccharomyces cerevisiae following expression of bar. In mammalian mitochondria, the mech anism of relocalization is thought to involve the opening of the so-called permeability transition pore. We show in this paper: (a) that bar-induced r elease of cytochrome c in yeast does not involve any permeability transitio n of the inner mitochondrial membrane but involves a general alteration of the permeability of the outer mitochondrial membrane to macromolecules. Thi s suggests that a permeability transition of the inner mitochondrial membra ne is not an event required for the relocalization of cytochrome c in yeast . (b) The outer-membrane voltage-dependent anion channel (VDAC), a putative component of the permeability transition pore, is not involved in bax-indu ced release of cytochrome c or in the prevention of this release by bcl-x(L ). (c) Bax devoid of its C-terminal putative hydrophobic alpha-helix is as efficient as full-length bar to allow the relocalization of cytochrome c, d emonstrating this segment of the protein is not required for membrane-targe ting. (d) We finally observe that the action of bar on the outer mitochondr ial membrane requires the presence of ATP both in vitro and in vivo, and it is shown that ATP directly increases the amount of bar inserted to mitocho ndria.