BCL-2 INHIBITS THE MITOCHONDRIAL RELEASE OF AN APOPTOGENIC PROTEASE

Bcl-2 belongs to a family of apoptosis-regulatory proteins which incor porate into the outer mitochondrial as well as nuclear membranes. The mechanism by which the proto-oncogene product Bcl-2 inhibits apoptosis is thus far elusive. We and others have shown previously that the fir st biochemical alteration detectable in cells undergoing apoptosis, we ll before nuclear changes become manifest, is a collapse of the mitoch ondrial inner membrane potential (Delta Psi(m)), suggesting the involv ement of mitochondrial products in the apoptotic cascade. Here we show that mitochondria contain a pre-formed similar to 50-kD protein which is released upon Delta Psi(m) disruption and which, in a cell-free in vitro system, causes isolated nuclei to undergo apoptotic changes suc h as chromatin condensation and internucleosomal DNA fragmentation. Th is apoptosis-inducing factor (AIF) is blocked by N-benzyloxycarbonyl-V al-Ala-Asp.fluoromethylketone (Z-VAD.fmk), an antagonist of interleuki n-1 beta-converting enzyme (ICE)-like proteases that is also an effici ent inhibitor of apoptosis in cells. We have tested the effect of Bcl- 2 on the formation, release, and action of AIF. When preventing mitoch ondrial permeability transition (which accounts for the pre-apoptotic Delta Psi(m) disruption in cells), Bcl-2 hyperexpressed in the outer m itochondrial membrane also impedes the release of AIF from isolated mi tochondria in vitro. In contrast, Bcl-2 does not affect the formation of AIF, which is contained in comparable quantities in control mitocho ndria and in mitochondria from Bcl-2-hyperexpressing cells. Furthermor e, the presence of Bcl-2 in the nuclear membrane does not interfere wi th the action of AIF on the nucleus, nor does Bcl-2 hyperexpression pr otect cells against AIF. It thus appears that Bcl-2 prevents apoptosis by favoring the retention of an apoptogenic protease in mitochondria.