Identification and characterization of two penta-EF-hand Ca(2+)-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 Ca(2+)-binding proteins that possess five EF-hand-like motifs. We identified two mutually homologous PEF proteins, designated DdPEF-1 and DdPEF-2 (64% amino acid residue identities), in the cellular slime mold Dictyostelium discoideum. Both 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 proteins) in the first EF-hand (EF-1) regions. Northern blot analyses revealed that DdPEF-1 and DdPEF-2 were constitutively expressed throughout development of Dictyostelium, but their levels of expression were developmentally regulated. In situ hybridization analyses demonstrated that DdPEF-1 was expressed 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 multicellular structures. Both PEF proteins were detected as 22-23-kDa proteins in soluble fractions in the presence of EGTA but in particulate fractions in the presence of Ca(2+) by Western blotting using specific monoclonal antibodies. Together 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 basic cellular functions.
Ohkouchi
S
S
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Nishio
K
K
Maeda
M
M
Hitomi
K
K
Adachi
H
H
Maki
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D002458:Cell Fractionation; D004023:Dictyostelium / Q000737:chemistry* / Q000235:genetics; D020832:EF Hand Motifs; D018507:Gene Expression Regulation, Developmental; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D010802:Phylogeny; D015800:Protozoan Proteins / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D002135:Calcium-Binding Proteins; C441461:PEF-1 protein, Dictyostelium discoideum; C441462:PEF-2 protein, Dictyostelium discoideum; D015800:Protozoan Proteins; D002118:Calcium