| Bcl-2 proteins regulate apoptosis in organisms as diverse as mammals and nematodes. These proteins are often localized at mitochondria by a C-terminal transmembrane domain. Although the transmembrane domain and mitochondrial localization are centrally involved in specific cases of vertebrate Bcl-2 activity, the significance of this localization is not clear for all species. Studying the C. elegans Bcl-2 homolog CED-9, we found that the transmembrane domain was both necessary and sufficient for localization at mitochondrial outer membranes. Furthermore, ced-9 transgenes lacking the transmembrane domain rescued embryonic lethality of ced-9 (lf) animals and responded properly to upstream signals in controlling the fate of Pn.aap neurons, although these transgenes were somewhat less active than equivalent transgenes derived from wild-type ced-9. Both of these apoptosic activities were retained in a construct where CED-9 lacking the transmembrane domain was targeted to the cytosolic surface of the endoplasmic reticulum and derived organelles, suggesting that in wild-type animals, accumulation at mitochondria is not essential for CED-9 to either inhibit or promote apoptosis in C. elegans. Taken together, these data are consistent with a multimodal character of CED-9 action, with an ability to regulate apoptosis through interactions in the cytosol coexisting with additional evolutionarily-conserved role(s) at the membrane.; Supporting a role for CED-9 at mitochondrial membranes, biochemical analysis revealed that CED-9 associates with membranes in manner similar to mammalian Bcl-2 proteins. Upon protonation, CED-9 lacking the transmembrane domain was able to bind to and remodel membrane vesicles. This membrane remodeling resulted in both membrane permeabilization and membrane fusion, suggesting a role for CED-9 at mitochondrial membranes.; One possible role may be to regulate mitochondrial dynamics. Over-expression of CED-9 in body wall muscles resulted in altered mitochondrial morphology, whereby long, tubular mitochondria seen in wild-type animals was converted to an interconnected, dilated network of mitochondria. These altered mitochondria can be partially suppressed by over-expression of the mitochondrial fission protein DRP-1, suggesting that this interconnected mitochondrial morphology may reflect a state of excess fusion. Taken together, these findings suggest that Bcl-2 proteins may localize at mitochondria to regulate mitochondrial dynamics. |
