- 學位論文
囊泡運輸參與胞質與粒線體間傳遞之功能探討
A Novel Vesicular-Transport Pathway Mediates the Uptake of Cytoplasmic Proteins into Mitochondria
摘要
過去研究指出老化相關疾病常伴隨粒線體形態及功能的異常，且年老的哺乳動物中，粒線體形態有巨大化的現象，顯示粒線體形態與功能的調控與老化息息相關，然而其中調控機制尚未清楚，因此我利用果蠅的肌肉組織為模型，探討粒線體於老化過程中形態及功能變化的詳細機制。我的研究中發現了參與調控老化的重要基因，Dosmit (Downsizing mitochondria)。Dosmit座落於粒線體外膜，其表現量隨老化漸增，並同時促進胞質蛋白進入粒線體，暗示此行為與老化相關，但機制仍未清楚。在Dosmit高量表現的條件下，粒線體呈現巨大化且內部產生許多含有胞質蛋白的雙層膜囊泡，而此囊泡被推測具有運輸功能。透過追蹤粒線體外膜蛋白Tom20，證明此雙層膜囊泡的膜系源自粒線體外膜。本論文進一步證明GTPase，Rab32不僅與Dosmit有蛋白間交互作用，且與Dosmit共同調控粒線體的巨大化及囊泡生成。於老化的果蠅中也可以觀察到粒線體巨大化及囊泡的產生，並發現泛素化蛋白大量在粒線體的囊泡中累積，且在粒線體蛋白酶Lon的缺失下，將引起更劇烈的泛素化蛋白累積現象，指出泛素化蛋白可能透過囊泡進入粒線體，並藉由蛋白酶Lon使其降解而維持粒線體功能平衡。Dosmit在哺乳動物中區分為Cisd1及Cisd2，而Dosmit卻融合了此二基因的序列，我將小鼠的Cisd1及Cisd2融合成重組基因，發現此重組基因能救回Dosmit剔除時所產生的粒線體缺陷。Dosmit的研究成果將為粒線體所造成的老化疾病提供新的治療途徑。
並列摘要
Mitochondrial aging, which results in mitochondrial dysfunction, is strongly linked to many age-related diseases. Aging is associated with mitochondrial enlargement and changes in the transport of cytosolic proteins into mitochondria. The underlying homeostatic mechanisms that regulate mitochondrial morphology and function, and breakdown during aging, remain unclear, though various important cytosolic proteins are known to be imported into mitochondria via a canonical pathway mediated by mitochondrial translocases (the TIM/TOM complex). Here I identify a novel pathway for mitochondrial protein trafficking in Drosophila melanogaster, involving the mitochondria-associated protein Dosmit. I found that Dosmit induces the formation of double-membraned vesicles within mitochondria, that the rate of vesicle formation increases with age, and that increased expression of Dosmit causes mitochondrial enlargement. When cytosolic green fluorescent protein (GFP) was ectopically expressed with Dosmit, intramitochondrial vesicles imported GFP from the cytosol. The outer-mitochondrial-membrane marker Tom20 was also associated with these vesicles, suggesting they originate from the outer mitochondrial membrane. The membrane trafficking process was mediated by the mitochondria-associated Rab protein Rab32. Dosmit expression levels were closely linked to the rate of aggregation of ubiquitinated proteins, which are themselves associated with age-related diseases. Finally, I found that expression of a hybrid form of the Dosmit mouse homolog resulted in a similar phenotype to that found with Drosophila Dosmit expression, suggesting a high level of evolutional conservation. This novel mitochondrial protein trafficking route mediated by Dosmit offers a promising target for future therapies targeting age-related mitochondrial diseases.