Abstract
Drosophila ECM is a CDGSH Ion-Sulfur binding domain containing protein that contains a CDGSH in C-terminal region. In this study, ECM is located on mitochondria at same direction. Overexpression of ECM dramatically causes Enlarged and Clustering Mitochondria in Drosophila S2 cell and mammalian HeLa cell so that we named it as ECM. We defined four regions in ECM, ECM-MH-1 and ECM-MH-2 from their similar and different region as N-terminal, transmembrane region, hinge region and CDGSH region. From the domain swap among ECM-MH-1, ECM-MH-2 and ECM, we found N-terminal of ECM is crucial for the mitochondrial enlargement and clustering in Drosophila and mammalian cell. ECM and ECM-MH-1 are located on mitochondria; the ECM-MH-2 is mainly located on ER in HeLa cell. We exchanged its transmembrane of ECM-MH-2 as the counter domain of ECM-MH-1 and ECM, this fusion ECM-MH-2 protein led to target to mitochondria and also trigger mitochondrial phenotype as same from ECM. We propose that N terminal domain of ECM-MH-2 and its Drosophila ortholog ECM has a critical function in the regulation of mitochondrial enlargement and clustering. In the live imaging, ECM promotes mitochondria to aggregate and cluster then cell divided in the end; it suggest this mitochondrial dynamics phenomenon might be regulated by cell cycle. Overexpression of ECM induced Drp1 to trans-locate on mitochondria and made mitochondria dull to dynamic. But this phenotype did not involve protein expression level of opa1 and drp1. We also found that anti-diabetes drug could reduce mitochondrial enlargement and clustering caused by ECM. However, which mechanism ECM is involved is not clear. By ECM-GFP fusion protein pull-down experiment, we found a protein, Hiw, is interacted with ECM by immunoprecipitation and LC/MS. Hiw, which is an ubiquitin E3 ligase, has been reported to be in the regulation of neuron growth. In this thesis, we evaluate which protein region of ECM that regulates the mitochondrial morphology change. With this clear understanding of ECM structure, function and genetic pathway, it might be possible to develop more therapeutic agents in ECM relative diseases, including aging and diabetes.