CED-4 is a key component of the central cell-death execution machinery.
ced-4, like
ced-3, is essential for programmed cell death (1). Recent biochemical studies show that CED-4 can bind to the Bcl-2 like protein CED-9 and the caspase CED-3 in vitro , in yeast and in mammalian cells (2,3,4). However, the physiological mechanism of CED-4 function remains unknown. To address this issue, we are studying the expression and subcellular localization of CED-4 in C. elegans. Immunostaining of whole-mount embryos with polyclonal antibodies raised against CED-4 protein reveals that CED-4 is expressed in most if not all cells during embryogenesis. In wild-type embryos, CED-4 expression exhibits a punctate, nucleus-excluded, web-like pattern, which is similar to the expression pattern of CED-9(B. Hersh and B. Horvitz, unpublished results). The CED-4 staining pattern overlaps partly, if not completely, with that of Mitotracker-Red, a mitochondria-specific marker. While the CED-4 expression pattern is not affected in
ced-3(lf) embryos, in
ced-9(lf);
ced-3(lf) embryos, CED-4 is no longer associated with mitochondria. Subcellular fractionation of worm embryos confirms our immunohistochemical results. Overexpression of EGL-1, a BH3(Bcl-2 Homology region 3)-containing cell-death activator in C. elegans(see abstract by B. Conradt and B. Horvitz), causes massive cell death in wild-type worms but not in
ced-9(
n1950gf) animals. We observe that EGL-1 overexpression induces the dissociation of CED-4 from mitochondria in wild-type worms but not in
ced-9(
n1950gf) worms. Our results suggest that CED-9 regulates the function and the subcellular localization of CED-4 in C. elegans by sequestering CED-4 onto mitochondria to prevent the killing function of CED-4. We postulate that the dissociation of CED-4 from CED-9 is necessary for the death process in dying cells and that this dissociation step is regulated by more upstream cell-death activators, such as EGL-1. 1. Ellis, H. and Horvitz, H.R. (1986). Cell 44, 817-829. 2. Wu, D., Wallen, H.D., and Nunez, G. (1997). Science 275, 1126-1129. 3. Chinnaiyan, A.M., O'Rourke, K., Lane, B.R. and Dixit, V.M. (1997). Science 275, 1122-1126. 4. Spector, M.S., Desnoyers, S., Hoeppner, D.J. and Hengartner, M.O. (1997). Nature 385, 653-656.