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Bcl-2 is a key inhibitor of a broad range of apoptotic pathways, yet neither the mechanism of action nor the role of Bcl-2 subcellular localization are well understood. The subcellular localization of Bcl-2 includes the mitochondrial membrane as well as the contiguous membrane of the endoplasmic reticulum and nuclear envelope. Most studies suggest that the ability of Bcl-2 to confer cell survival is dependent upon its localization to the mitochondria. In this manuscript, we show that Bcl-2 targeted to the endoplasmic reticulum can inhibit Myc-, but not etoposide-induced apoptosis in the Rat-1 fibroblast cell line. By contrast, wild type Bcl-2 can inhibit apoptosis triggered by either death agonist. We further show both Myc and etoposide trigger disruption of mitochondrial membrane potential (MMP) and induce poly-ADP ribose polymerase (PARP) cleavage, but release of calcium was not evident. Bcl-2 abrogates apoptosis at or upstream of MMP depletion showing that Bcl-2 does not have to reside at the mitochondria to prevent apoptosis. These results further elucidate the biochemical events associated with Myc- and etoposide-induced apoptosis and significantly advance our understanding of Bcl-2 function.

Human Bcl-2 is located in multiple intracellular membranes when expressed in MDCK and Rat-1/myc cells. We restricted expression to the endoplasmic reticulum or mitochondria by exchanging the Bcl-2 carboxy-terminal insertion sequence for an equivalent sequence from cytochrome b5 or ActA, respectively. MDCK cells are protected from serum deprivation-induced apoptosis by both wild-type Bcl-2 and the mutant targeted to mitochondria but not by the mutant targeted to endoplasmic reticulum. In contrast, when expressed in Rat-1/myc cells, the Bcl-2 mutant located at the endoplasmic reticulum is more effective than that targeted to mitochondria. In MDCK cells both mutants bind Bax as effectively as wild-type, demonstrating that Bax binding is not sufficient to prevent apoptosis.