探討Atg20在調控選擇性細胞自噬中所扮演的角色

The role of Atg20 in the regulation of selective autophagy pathways

謝瑩蓉(Ying-Jung Hsieh)

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(2011年)

https://doi.org/10.6342/NTU.2011.02879

摘要

細胞自噬（autophagy）是細胞分解大分子物質的途徑之一，在真核細胞中有高度的保守性。其作用過程透過新生成的雙層膜自噬體（autophagosome）構造包覆細胞質中的大分子物質或整個胞器，接著自噬體的外膜與液胞或溶酶體（vacuole/lysosome）進行融合，將自噬體的內膜與內容物分解，所產生的基本組成單位則用以維持細胞體內的恆定。在酵母菌（Saccharomyces cerevisiae）研究系統中發現，有兩種選擇性的細胞自噬採取了類似的運作機制：其一為分解細胞內的過氧化小體（peroxisome），稱為過氧化小體自噬途徑（pexophagy）；其二是運送液胞水解酵素aminopeptidase 1 前驅物（prApe1）、α-mannosidase 1（Ams1）以及aspartyl aminopeptidase (Ape4)至液泡，此途徑稱為細胞質至液泡傳遞途徑（cytoplasm-to-vacuole targeting pathway, Cvt pathway）。其中過氧化小體自噬體（pexophagosome）和細胞質至液胞運輸小泡（Cvt vesicle）的雙層膜構造並非直接來自已存在的膜，而是重新組合而成，其生成過程目前仍有諸多機制不明。

已知Atg20 參與在這兩種途徑且與細胞自噬的核心複合體有直接的交互作用，其具有PX 功能性區域（PX domain）與PtdIns(3)P（phosphatidylinositol 3-phosphate）結合，此交互作用對於Atg20 在細胞質中的位置以及執行功能扮演著重要的角色。本篇論文指出，Atg20 參與在細胞質至液胞運輸小泡的形成過程當中，同時針對Atg20 的兩個螺旋捲曲功能性區域（coiled-coil domain）進行探討。結果顯示當此兩個功能性區域被去除時，所造成的缺失包含，運輸小泡無法正常形成以及早期的細胞自噬蛋白—Atg1、Atg9 和Atg11 累積在自噬體前驅構造(PAS, pre-autophagosomal structure）。進一步的研究更發現，雖然這兩個功能性區域的缺失對於Atg20 與已知有交互作用的細胞自噬蛋白—Atg11、Atg17 和Atg24 的結合沒有影響，但卻會使得Atg20 和Atg24 出現在PAS 的現象受到影響。研究也發現除了PtdIns(3)P 外，Atg24 和Trs85 對於Atg20 運輸到PAS 也扮演了重要的角色，因此我們假設三者可能共同調控運輸小泡的生成。另一方面，我們利用螢光顯微鏡觀察Snc1 在細胞當中的位置，發現Atg20 螺旋捲曲功能性區域的缺失只會造成部分Snc1 無法從內質體（endosome）運輸到高基氏體（Golgi apparatus），顯示細胞質至液胞傳遞途徑的嚴重缺損並非由失去功能的內膜傳遞系統所造成。此外，Atg20 螺旋捲曲功能性區域缺失也會造成過氧化小體自噬途徑無法正常執行。綜合以上結果，我們認為Atg20 的兩個螺旋捲曲功能性區域對於調控細胞質至液胞運輸小泡的形成扮演著重要的角色，且這個過程需要Atg24和Trs85 的共同參與。

關鍵字

細胞質至液泡傳遞途徑 ； 過氧化小體自噬途徑 ； 細胞質至液泡運輸小泡之形成 ； Atg20

並列摘要

Autophagy is an evolutionarily conserved catabolic pathway in eukaryotic cells, which mediates the turnover of cytosolic components sequestered by double membrane vesicles and delivered to the lysosome/vacuole to be degraded to maintain cellular homeostasis. Saccharomyces cerevisiae has adapted this mechanism for delivery of peroxisome by pexophagy or transport specific vacuolar hydrolases, including aminopeptidase I (Ape1), α-mannosidase (Ams1), and aspartyl aminopeptidase (Ape4), via the cytoplasm-to-vacuole targeting (Cvt) pathway. The mechanism underlying the biogenesis of the Cvt vesicle and pexophagosome are barely understood.

Atg20 is a Cvt pathway-specific component with a conserved phox homology domain (PX domain) involved in binding to phosphatidylinositol 3-phosphate [PtdIns(3)P], and this binding ability is necessary for its distribution and function. Here, I show that Atg20 participates in the Cvt vesicle formation, and this process is dependent on its two coiled-coil domains. Since the Cvt pathway is blocked in atg20 mutant cells maintained in growing conditions, early-action Atg proteins, including Atg1, Atg9, and Atg11, are seen accumulated at the pre-autophagosomal structure (PAS). Detailed analyses showed that deletion of the coiled-coil domains of Atg20 abolishes the PAS localization of Atg20 and Atg24, while the interaction between Atg20 and its known interacting partner, Atg11, Atg17, and Atg24, is not affected. Furthermore, the PAS localization of Atg20 requires not only its PtdIns(3)P-binding ability but also the presence of Atg24 and Trs85, which is also required for the formation of the Cvt vesicle, indicating they may function together for the completion of the Cvt vesicle via unknown mechanism. In addition, Atg20 belongs to the sorting nexin (SNX) family, which is involved in the retrieval of proteins from the early endosomes to the late Golgi apparatus. Mutation of atg20, however, only causes partial defects in the retrieving of Snc1, suggesting that the Cvt pathway defect is not due to the disruption of the endomembrane trafficking. Moreover, I also find deletion of the coiled-coil domains of Atg20 causes pexophagy defect. Together, it is concluded that the coiled-coil domains of Atg20 are essential for the Cvt vesicle formation in cooperation with Atg24 and Trs85 under vegetative growth conditions.

並列關鍵字

Cvt pathway ； pexophagy ； Cvt vesicle formation ； Atg20

參考文獻

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