Mapping of the interaction sites between apoptosis linked gene ALG-2 and HEED

The apoptosis linked gene (ALG-2) is a 22 kDa Ca 2 + -binding protein of the penta EF-hand motif family. ALG-2 was discovered in a "death trap" assay using T-cell receptor-mediated apoptosis. Depletion of ALG-2 using an anti-sense ALG-2 construct inhibits apoptosis that is induced by several stimuli, such as staumsporin, dexamethason, Fas, and glucocorticoid. The Ca 2 + -dependent function of ALG-2 is consistent with the observation that the cytoplasmic Ca 2 + level is elevated in apoptotic cells. We found that ALG-2 interacted specifically with the carboxy-terminal region of HEED using a yeast two-hybrid assay system. The mutants of HEED were constructed by deleting five WD repeat motifs one by one from the C-terminus of the protein. These mutants of ALG-2 were made by combining the EF hand Ca 2 + binding motifs in various ways. Mapping of the interaction sites, using each of the mutants, revealed that the interaction between HEED and a third EF-hand motif of ALG-2 was stronger than the other combination.