Expression and characterization of a Dictyostelium discoideum annexin.
The annexins are calcium-dependent phospholipid-binding proteins. Recently the gene encoding the homologue of a mammalian annexin has been identified in Dictyostelium discoideum. Analysis of cDNA and genomic clones showed that the transcript for Dictyostelium annexin is alternatively spliced (Greenwood, M. and Tsang, A. (1991) Biochim. Biophys. Acta 1088, 429-432; Döring, V., Schleicher, M and Noegel, A. (1991) J. Biol. Chem. 266, 17509-17515). Here, we showed that the Dictyostelium annexin DNA hybridized to two populations of transcripts. We used a recombinant annexin polypeptide to raise polyclonal antibody. Immunoblot analysis revealed that the antibody recognized two polypeptides of 48 kDa and 54 kDa in developing D. discoideum cells. The molecular sizes of these polypeptides correspond well with the expected sizes of the alternatively spliced products. The 48-kDa and 54-kDa polypeptides were purified by isoelectric focusing to more than 70% homogeneity. The partially purified proteins were found to associate with phosphatidylserine vesicles in a calcium-dependent manner. These results suggest that the 48- and 54-kDa polypeptides are the products of alternative splicing of the annexin transcripts. During development the two polypeptides accumulate at different rates to about 60 times the level detected in vegetative cells. On the other hand, RNA blot analysis showed that the level of the annexin transcripts in multicellular aggregates was about 5 times that of vegetative cells.
Bonfils
C
C
Department of Biology, Concordia University, Montreal, Quebec, Canada.
Greenwood
M
M
Tsang
A
A
D017398:Alternative Splicing / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D017302:Annexins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D018076:DNA, Complementary; D004023:Dictyostelium / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D008081:Liposomes / Q000378:metabolism; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000378:metabolism; D011110:Polymorphism, Genetic / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins
D017302:Annexins; D018076:DNA, Complementary; D008081:Liposomes; D010743:Phospholipids; D011993:Recombinant Fusion Proteins; D002118:Calcium