建立細胞自噬蛋白交互辨識圖譜與分析運輸蛋白Trs23和Trs85在細胞自噬上扮演之角色

Construction of the Atg Protein Interaction Map and Study of the Roles of Trs23 and Trs85 in Autophagy pathway

蔡怡馨(Yi-Shin Tsai)

指導教授 ： 黃偉邦

國立臺灣大學/生命科學院/動物學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.01669

摘要

細胞自噬（autophagy）途徑為真核生物特有的膜運輸（membrane trafficking）機制，它對於單一細胞的存活扮演重要的角色。在養分缺乏的環境下，細胞自噬可以扮演維持生命的功能，這個機制是透過一個雙層膜的自噬體（autophgosome），包裹一些細胞質中的物質和胞器，透過與溶小體或液泡（lysosome/vacuole）癒合而完成消化分解作用，以提供細胞能量來源並維持細胞生存。
本篇論文的第一部份，透過酵母菌雙雜交實驗分析，建立細胞自噬蛋白交互辨識圖譜，並與已知的細胞自噬蛋白質間的交互作用資訊分析，深入探討細胞自噬的分子調控機制。
本篇論文的第二部份，則是透過酵母菌雙雜交實驗，尋找酵母菌的cDNA庫中可與細胞自噬蛋白Atg9作用的分子；由此實驗中所發現Atg9交互作用的17個蛋白中，選擇Trs23深入探討其與細胞自噬間的關係。Trs23與Trs85為TRAPP複合體的成員，其中Trs85已被證實參與調控細胞自噬及細胞質至液泡傳遞途徑（cytoplasm to vacuole targeting, Cvt pathway），因此我進一步分析Trs23是否與細胞自噬或Cvt途徑的調控有關。另外，Trs85如何調控細胞自噬以及細胞質至液泡傳遞途徑至今仍不清楚，因此我也嚐試測試Trs85與Atg9分子直接交互作用的可能性。

關鍵字

細胞自噬 ； 自噬體 ； TRAPP蛋白質複合 ； 酵母菌雙雜交系統 ； 交互作用圖表

並列摘要

Autophagy is a membrane-trafficking mechanism conserved in all eukaryotic cells, which plays an important role for survival of unicellular organisms in nutrient starvation conditions. During the starvation conditions, a double membrane-bound vesicle, named an autophagosome, mediates the degradation of cytosolic components in the lysosome/vacuole to provide energy sources.
For the first part of this study, I used the dual reporter yeast two-hybrid system to establish the Atg protein interaction map. Comparing to already published Atg protein interactions, this Atg protein interaction map may bring us insights for future analysis of the autophagy molrculer mechainsm.
For the second part of this study, I used Atg9 as a bait protein to screen for it’s interaction partners. Among those 17 proteins identified by the yeast two-hybrid analysis as Atg9 interaction proteins, Trs23 is particularly interesting and was chosen for further study. Like Trs85, Trs23 is a subunit of the TRAPP complex, which regulates membrane trafficking between the ER and the Gogi complex. Trs85 has already been reported to be required for the autophagy and Cvt pathway. Therefore, I was interested to test if Trs23 was required for the autophagy and Cvt pathway or not.
In addition, the detailed molecular mechanism underlying how Trs85 regulates autophagy and the Cvt pathway is still unknown. I have examined if Trs85 like Trs23 also interact with Atg9 to facilitate autophagic transport.

並列關鍵字

autophagy ； Trs85 ； TRAPP ； autophagosme ； yeast two hybrid

參考文獻

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