| Mitochondria, double membrane-bounded organelles in eukaryotic cells, are dynamic organelles constantly undergoing fusion and fission, and play an important role in energy production, apoptosis and aging. Regarding mitochondrial fusion, several protein molecules have been reported to mediate this process. Given the involvement of acidic phospholipids in SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) complexes-mediated vesicle fusion, however, it is reasonable to speculate that those phospholipids are also components of mitochondrial fusion machinery. Regarding apoptosis, cardiolipin is a dimeric phospholipid only found in bacterial and mitochondrial membranes and regulates mitochondrial apoptotic pathway via cytochrome c release and tBid translocation. Although most of the apoptosis research has been carried out in mammalian cells, the enzyme synthesizing cardiolipin, cardiolipin synthase, has not been cloned yet in animals.; For my thesis, I have isolated and characterized Mito-PLD, a novel member of the PLD superfamily. Mito-PLD localizes to an outer mitochondrial membrane and, when overexpressed, induces mitochondrial aggregation, which is dependent on a Mito-PLD's HKD motif, a signature motif of the PLD superfamily. Knockdown of Mito-PLD by RNA interference (RNAi) fragments mitochondria. Mito-PLD functions as a lipid modifying enzyme acting on phospholipids of adjacent mitochondria (that is, acts in trans) and mediates mitochondrial fusion, which is dependent on mitofusin, an outer mitochondrial membrane protein tethering mitochondria. Ablation of Mito-PLD in Drosophila melanogaster alters fused mitochondrial morphology during spermatogenesis. These results suggest that Mito-PLD is a mitochondrial lipid modifying enzyme mediating mitochondrial fusion. Location of Mito-PLD in the chromosomal region deleted in Smith-Magenis syndrome suggests that perturbation of Mito-PLD function might result in human pathogenesis.; In addition, I have also isolated and characterized human cardiolipin synthase (hCLS). Human CLS localizes to mitochondria and overexpression of hCLS complements phenotypes of cls null Saccharomyces cerevisiae. Cell lines harboring reduced level of hCLS through RNAi have been established and a study on the effect of hCLS knockdown on cytochrome c release and tBid translocation is underway. Barth syndrome is an X-linked recessive genetic disorder featuring a triad of dilated cardiomyopathy, skeletal myopathy and neutropenia, and caused by a decrease in cardiolipin level. Overexpression of hCLS in Barth syndrome patients could be a therapeutic modality to restore normal cardiolipin levels. |
