Identification And Functional Analysis Of A Novel Gene Ecp In Drosophila

The cDNA [GenBank accession No.AF325529] of ecp was cloned from Drosophila embryonic brain cDNA library. Little is known about the function of ecp till Josh Dubnau reported that P-element insertion mutant line C0150 of Drosophila defected in long term memory which indicate it may be involved in learning and memory. ecp mRNA is 1572 bp in length and contains a complete ORF encoding a protein of 422 amino acids. Homologs of Ecp exist in various organisms from Danio rerio, xenopus laevis to mammalians. Prediction by using PROSITE shows that Ecp contains an N-terminal leucine zipper motif (amino acids 39-60) and a C-terminal eIF5C domain (amino acids 327-406), which was responsible for the binding of eIF5 with the K domain of eIF2β. The high similarity of the eIF5C domain between ECP and eIF5 suggests ECP might also interact with eIF2β.In order to analyze function of ECP, monoclonal and polyclonal antibodies against ECP were generated, and both could specifically detect recombinant ECP from bacterial and endogenous ECP. The results of Drosophila embryo immunostaining using these antibodies show that ecp expressed ubiquitously in Drosophila embryos, which was consistent with that of embryo whole mount RNA in situ hybridization. Western blotting results also indicated that ecp expressed continuously throughout Drosophila development from the embryo to adult. Both GFP tagged ECP in cell line HEK293 and endogenous ECP in Drosophila S2 cells and embryos were predominantly distributed in the cytoplasm. These results are consistent with the predicted function of ECP to be involved in protein synthesis through interaction with eIF2β.Two isoforms of ECP could be detected in Drosophila by Western Blotting. In embryo, pupa and adult stage, the slow mobility isoform (ECP-S) is the predominant one while the fast mobility isoform (ECP-F) is the main one expressed in the first and second instar larva. Moreover, ECP-F substitutes the slow one in embryos immediately after the hatching of the eggs. On the other hand, ECP-F gradually shifts back to ECP-S when the third instar larvae starts to form pupae. The periodical shifts of the 2 isoforms during Drosophila development indicate that ecp might be related to the larva growth.The results from mass spectrum show that many ribosomal proteins including Drosophila RPL5 exist in the immunoprecipitated complex formed by monoclonal antibody against ECP. In addition, Ecp cosediments with both 40S and 60S ribosomal subunits on sucrose gradient. These results indicate that Ecp might be a ribosome-associated protein. eIF2βand RPL5 were found to interact with ECP in a Y2h screening. The interaction of ECP and RPL5 was confirmed by GST pulldown and coIP. Moreover, it was showed that the fragment (1–150) which contained the predicted leucine zipper motif was sufficient for its binding with RPL5.The above results support ecp might play some role in protein synthesis. Actually, incorporation of S35-Methionine decreased by 45% compared to control after knockdown of ECP by RNAi in S2 cells. Besides, typical minute phenotypes of short bristles, slow development and...