Tagging of C. elegans apoptosis activator EGL-1 BH3-only reveals CED-9 BCL-2-dependent mitochondrial localization and dynamic control of EGL-1 synthesis and degradation in vivo

The BH3-only protein EGL-1 is the key activator of apoptosis during C. elegans development. EGL-1 protein is thought to be synthesized predominantly in cells programmed to die and to localize to mitochondria. We used CRISPR-Cas-mediated modification of the egl-1 locus to add the coding sequence for the monomeric StayGold fluorescent protein or 18 copies of the SunTag peptide to the endogenous open reading frame. We found that tagged EGL-1 protein colocalizes with mitochondria in vivo and that mitochondrial localization is dependent on the anti-apoptotic BCL-2-like protein CED-9. Consistent with the presence of egl-1 mRNA in cells programmed to die as well as their progenitor cells ('mother' cells), EGL-1 protein is detected in both types of cells in vivo. Furthermore, real time imaging reveals that EGL-1 protein rapidly disappears from the mother cell prior to its division and that EGL-1 protein rapidly reappears specifically in the daughter cell programmed to die. Our results demonstrate CED-9 BCL-2-dependent mitochondrial localization of EGL-1 BH3-only protein and dynamic control of EGL-1 protein synthesis and degradation. Furthermore, we have identified additional levels of control of egl-1 BH3-only function that expand our understanding of apoptosis activation in vivo.