Alterations in mitochondrially localized Bcl-2/Bax interactions in intact cells during apoptosis

ApoptoMn is an evolutionary conserved physiological process of tell death iinolving a cascade ot characteristic biochemical and iillratnutiiral events. Proteins of Bcl-2 family play a crucial role not only in controlling ecu death but also in initiation and progression of human cancers. It has been .shown thai the e|e\uted expression of Bcl-2 protein prevents apoptosis induced b> n wide variety of death-inducing stimuli, while Max which belongs to the Bel 2 family, promotes apoptosis when overexpressed. It has been hypothesi/ed that Bel 2 and H ax form both hetro- and homodimers and that the relative ratio of Bcl-2/Bax hetro- versus homodimers regulates apoptosis. To date, howevei. the interaction of Bcl-2 and Bax at the intact cell level or changes in their inter at t ion. in relation to the apoptotic response have not been demonstrated. Here \\e show Bcl-2 and Bax interact with each other in intact mammalian cells. The interaction of green fluorescent protein (GFP) Bax with blue fluorescent protein ( B h'I') Bcl-2 was documented using fluorescence resonance energy transfer (FRET) after expression of these two oncoproteins in the same cells as (IFF and Br-P fusion proteins, respectively. Further, we demonstrate that this in teraction in vivo takes place in mitochondria where Bcl-2 and Bax proteins are known to reside. The effect of Taxol. Atractyloside, FCCI'/CCCF. Mitom>cni (' and Cisplatin on changes in the Hax-BcI-2 interaction (measured by i hinges in FRF'F), is being studied. To our knowledge this is first demonstra lion of protein-protein interaction in mammalian cells using two independent flunrescriiily labeled endogenous!)- expressed fusion proteins.