Distinct roles of mitochondria- and ER-localized Bcl-xL in apoptosis resistance and Ca2+ homeostasis.

Bcl-2 proteins are major regulators of cellular responses to intrinsic and extrinsic apoptotic stimuli. Among them, overexpression of the antiapoptotic protein Bcl-xL modulates intracellular Ca2+ homeostasis and organelle-specific apoptotic signaling pathways. However, the specific activities of Bcl-xL at mitochondria and the endoplasmic reticulum (ER) have not been fully defined. To further explore this, we generated mouse embryonic fibroblast (MEF) cell lines deficient in Bcl-xL expression (Bcl-x-KO). Deficiency in Bcl-xL expression did not induce compensatory changes in the expression of other Bcl-2 proteins, and Bcl-x-KO MEF cells showed increased sensitivity to various apoptotic stimuli compared with wild-type MEF cells. Targeting Bcl-xL at mitochondria but not at the ER restored apoptosis protection in Bcl-x-KO MEF cells to the degree observed in wild-type MEF cells. However, expression of ER-targeted Bcl-xL but not mitochondrially targeted Bcl-xL was required to restore Ca2+ homeostasis in Bcl-x-KO MEF cells. Of importance, ER-targeted Bcl-xL was able to protect cells against death stimuli in the presence of endogenous Bcl-xL. These data indicate that mitochondrial Bcl-xL can regulate apoptosis independently of ER Bcl-xL and that when localized exclusively at the ER, Bcl-xL impinges on Ca2+ homeostasis but does not affect apoptosis unless Bcl-xL is present in additional cellular compartments.