Identification and characterization of two penta-EF-hand Ca+ binding proteins in Dictyostelium discoideum

Penta-EF-hand (PEF) proteins such as ALG-2 (apoptosis-linked gene 2 product ) and the calpain small subunit are a newly classified family of Ca2+-bindi ng proteins that possess five EF-hand-like motifs. We identified two mutual ly homologous PEF proteins, designated DdPEF-1 and DdPEF-2 (64% amino acid residue identities), in the cellular slime mold Dictyostelium discoideum. B oth PEF proteins showed a higher similarity to mammalian ALG-2 and peflin ( Group I PEF proteins) than to calpain and sorcin subfamily (Group II PEF pr oteins) in the first EF-hand (EF-1) regions. Northern blot analyses reveale d that DdPEF-1 and DdPEF-2 were constitutively expressed throughout develop ment of Dictyostelium, but their levels of expression were developmentally regulated. In situ hybridization analyses demonstrated that DdPEF-1 was exp ressed in both the anterior prestalk and the posterior prespore regions of the tipped aggregate, slugs and early culminants. On the other hand, DdPEF- 2 was dominantly expressed in the anterior tip region of these multicellula r structures. Both PEF proteins were detected as 22-23-kDa proteins in solu ble fractions in the presence of EGTA but in particulate fractions in the p resence of Ca2+ by Western blotting using specific monoclonal antibodies. T ogether with the finding of PEF-like sequences in DNA databases of plants, fungi and protists, our results strongly suggest that Group I PEF proteins are ubiquitously present in all eukaryotes and play important roles in basi c cellular functions.