A conserved N-terminal sequence targets human DAP3 to mitochondria

Human DAP3 (death-associated protein-3) has been identified as an essential positive mediator of programmed cell death. Structure-function studies hav e shown previously the N-terminal extremity of the protein to be required i n apoptosis induction. Analysis of human DAP3 gene structure predicted 13 e xons and subsequent targeting prediction by two software packages (MITOPROT and TargetP) gave a high probability for mitochondrial targeting. The pred icted N-terminal targeting structure was also found in the mouse, Drosophil a, and C. elegans orthologues with a strong sequence homology between mouse and human. Secondary structure analyses identified ar-helical structures t ypical of mitochondrial target peptides. To confirm experimentally this tar geting we constructed a fusion protein with N-terminal human DAP3 upstream of enhanced green fluorescent protein (EG;FP). Confocal analysis of transfe cted human fibroblasts clearly demonstrated EGFP localization exclusive to mitochondria. The positioning of this key apoptotic factor at the heart of the mitochondrial pathway provides exciting insight into its role in progra mmed cell death. (C) 2001 Academic Press.