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  • 學位論文

探討Gyp1於自噬作用中之角色

The role of Gyp1 in autophagy regulation

指導教授 : 黃偉邦

摘要


細胞自噬作用(Autophagy)是一個具有高度演化保守性的細胞反應機制。為了應對細胞外部的各種環境壓力、細胞內部養分的缺乏或是胞器與蛋白質的代謝需求,細胞會啟動各種不同類型的自噬作用以維持細胞生存、能量供給或是生理上的適應。細胞自噬作用受到高度的調控,許多不同的蛋白參與其過程,其中有一部分蛋白質參與各種類型自噬作用的執行,被稱為核心自噬作用蛋白(core autophagy related proteins),而這些蛋白可以被簡單區分成不同的單白質複合物(protein complex),其中包括Atg1蛋白質複合物。Atg1蛋白質複合物參與在早期的自噬作用調控機制,當自噬作用被啟動之後,Atg1蛋白質複合物可以迅速的被轉移到自噬作用的啟動位置上,並且輔助其他蛋白組成骨架以利後續自噬體(autophagosomes)的形成,而當自噬體完整形成後,Atg1複合體可以一併送往濾泡(Vacuole)分解,抑或是重新循環回細胞質中再利用。 除了核心自噬作用蛋白之外,還有許多不同的機制參與在整個自噬作用的過程中,例如為了供給形成自噬體過程中所需大量的脂質來源,需要細胞內部內膜運輸機制的配合。為了能妥善調控內膜運輸的機制,需要非常多蛋白複合體的參與,其中就包括了控制細胞內膜運輸機制的GTP酶-Rab/Ypt/Sec家族(Rab/Ypt/Sec protein family)。在Rab/Ypt/Sec家族中,有數種蛋白在先前的研究報告中已經討論過其在自噬作用中所扮演的角色,而在其中Ypt1以及Ypt7被認為是控制早期自噬作用非常重要的蛋白酶,為了精密的控制GTP酶在細胞中的作用,GTP酶的活性會受到GTP酶活化蛋白(GTPase activation protein)的調控,在其中Gyp1被認為是細胞中調控Ypt1以及Ypt7的重要蛋白。本研究中,我們發現缺乏GTP酶活化蛋白Gyp1的細胞會出現細胞自噬的缺陷,且此種缺陷在不同品系的出芽酵母菌株中出現,除此之外,缺乏Gyp1也會影響選擇性自噬作用,並使得Atg1蛋白停留在自噬體形成位置(preautophagosomal structure)上的時間增加。我認為當細胞缺乏Gyp1時會導致細胞內部內膜運輸機制失衡,進一步延緩細胞內部自噬體的生成,造成細胞自噬功能受到部分的影響。

並列摘要


Autophagy is a highly conserved catabolism pathway responding to nutrient starvation, stresses, and extracellular signals. It is a precisely regulated process, which defect is related to many human diseases. In order to cope with different cellular stresses, cells could elicit different types of autophagy, including nonselective autophagy and selective autophagy. Many protein complexes are involved in executing these different types of autophagy. Among them, Atg1 is recruited to the PAS at the early stage of autophagy and forms the basic scaffold structure for autophagosome formation. After the formation of autophagosomes, the completed autophagic vesicles fuse with the vacuole and complete the whole pathway. In addition to the core autophagy regulatory proteins, there are other important factors indispensable for autophagy execution. In the whole autophagy process, the formation of autophagosome would rely heavily on membrane trafficking. Membrane trafficking is an essential process in cell physiology, and is regulated by Rab/Ypt protein family. The Rab/Ypt proteins are GTPases whose activities are modulated by GTPase activation proteins (GAPs) and Guanine nucleotide exchange factors (GEFs). In yeast, Ypt1 and Ypt7, which are Rab/Ypt proteins known to mediate the early and late autophagy process, respectively. In this study, I found that deletion of GYP1, which is a GAP protein of Ypt1 and Ypt7 in yeast, would cause autophagy defect. The autophagy defect of gyp1∆ cells is conserved among different yeast strains and is correlated to viability decrease of the mutant cells under nitrogen starvation stress. Deletion of GYP1 would further cause minor Cvt pathway defect and moderate pexophagy defect. Although I could not observe any change of Atg protein modification in gyp1∆ cells comparing to wild type cells, higher percentages of gyp1∆ cells do show Atg1 puncta at the PAS. I suppose that deletion of GYP1 would disrupt the dynamic balance of membrane trafficking and delay the membrane supply for autophagosomes formation. In conclusion, our results indicated that Gyp1 is required for the control of normal Atg1 recycling, indicating the importance of maintaining the dynamic movement of the endomembrane system in autophagy regulation.

並列關鍵字

Autophagy Atg1 Gyp1 Rab family proteins

參考文獻


Kirisako, Takayoshi, Misuzu Baba, Naotada Ishihara, Kouichi Miyazawa, Mariko Ohsumi, Tamotsu Yoshimori, Takeshi Noda, and Yoshinori Ohsumi. "Formation Process of Autophagosome Is Traced with Apg8/Aut7p in Yeast." The Journal of cell biology 147, no. 2 (1999): 435-46.
Mizushima, Noboru, Takeshi Noda, Tamotsu Yoshimori, Yae Tanaka, Tomoko Ishii, Michael D George, Daniel J Klionsky, Mariko Ohsumi, and Yoshinori Ohsumi. "A Protein Conjugation System Essential for Autophagy." Nature 395, no. 6700 (1998): 395-98.
Albert, S., and D. Gallwitz. "Two New Members of a Family of Ypt/Rab Gtpase Activating Proteins. Promiscuity of Substrate Recognition." J Biol Chem 274, no. 47 (Nov 19 1999): 33186-9.
Albert, Štefan, Elke Will, and Dieter Gallwitz. "Identification of the Catalytic Domains and Their Functionally Critical Arginine Residues of Two Yeast Gtpase‐Activating Proteins Specific for Ypt/Rab Transport Gtpases." The EMBO Journal 18, no. 19 (1999): 5216-25.
Bacon, Rebecca A, Antti Salminen, Hannele Ruohola, Peter Novick, and Susan Ferro-Novick. "The Gtp-Binding Protein Ypt1 Is Required for Transport in Vitro: The Golgi Apparatus Is Defective in Ypt1 Mutants." The Journal of Cell Biology 109, no. 3 (1989): 1015-22.

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