Atg20的功能性區域與其在細胞質至液胞傳遞途徑中的機制

Insight into Atg20: Its functional domains and the mechanism in the Cvt pathway

陳昀伶(Yun-Ling Chen)

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(2013年)

https://doi.org/10.6342/NTU.2013.10589

摘要

細胞自噬（autophagy）一直被認為是一種非選擇性分解大分子物質的機制，在養份缺乏的環境下，藉此維持細胞內的恆定性與生存，且在真核生物中具高度保守性。然而近年的研究發現，在出芽酵母菌細胞中存在著另一種具選擇性的機制，稱為細胞質至液胞傳遞途徑（cytoplasm-to-vacuole targeting pathway, Cvt pathway）。在養分充足的情況下，此途徑會將液胞水解酵素aminopeptidase1前驅物（prApe1）、α-mannosidase1（Ams1）以及aspartyl aminopeptidase（Ape4）運送至液胞。雖然前人的文獻已報告Atg20參與在細胞質至液胞傳遞途徑中，但其詳細的作用機制還不甚明瞭。此外，已知Atg20具有一個phox homology domain（PX domain），此PX domain 能專一性的與PI3P結合並藉此決定了Atg20在細胞中的分布，但此結合的親和性與其他同樣具有PX domain的蛋白質比較起來相對偏低，顯示可能有其他協助Atg20在細胞中分布的機制存在。本研究發現，Atg20蛋白的BAR（Bin/Amphiphysin/Rvs）結構性區域中的小片段缺損，會造成細胞質至液胞傳遞途徑無法完成以及內噬系統的運送缺失。進一步分析則發現，BAR結構性區域中的長段雙極性螺旋（Amphipathic helix L, AHL）以及其最末端49個胺基酸對於Atg20與Atg24移動到自噬體形成位置（pre-autophagosomal structure, PAS）以及Snc1在內噬系統中的回收是必須的。而失去了最後49胺基酸的Atg20更喪失了與Atg24的結合能力，並造成了減緩細胞增生速度的後果。另外，同時定點突變位於AHL中的Leu378和Ile382雖然不影響Atg20與膜的結合能力，但卻會使運輸小泡（Cvt vesicles）無法正常形成。最後，Atg20與Atg24須藉由Ymr1的幫助將PI3P去磷酸化後，才得以自PAS被釋回細胞質中。綜合以上所述，BAR結構性區域藉由不同的功能，影響細胞質至液胞傳遞途徑以及內膜傳遞系統（endomembrane trafficking）。此外，Atg20以及Atg24在運輸小泡形成後會從PAS被回收再利用。

關鍵字

細胞自噬 ； 細胞質至液胞傳遞途徑 ； Snc1回收 ； Atg20 ； BAR區域 ； 雙極性螺旋 ； 細胞質至液胞運輸小泡的形成

並列摘要

Autophagy has been considered as a non-selective degradation process in order to maintain cellular homeostasis upon starvation for a long time. This physiological response is highly conserved in all eukaryotes. Recent studies have found that the cytoplasm-to-vacuole targeting (Cvt) pathway of the budding yeast is a selective type of autophagy, which constitutively transports prApe1, Ams1, and Ape4 to the vacuole under vegetative growth condition. Although Atg20 has been reported required for the Cvt pathway, how it participates in the control of the Cvt pathway remains largely unknown. Recent studies have shown that Atg20 contains a conserved phox homology domain (PX domain), which could specifically bind to PI3P for determining the distribution in the cells, but the affinity is relatively low. Here, I found that deletion of the Bin/Amphiphysin/Rvs (BAR) domain of Atg20 impairs its ability to regulate the Cvt pathway and endomembrane trafficking. I further showed that the long amphipathic helix (AHL) and the last 49 residues of Atg20 are essential for the localization of Atg20 and Atg24 to the pre-autophagosomal structure (PAS) and for the retrieval of Snc1 from the endocytic pathway. Moreover, lacking the last 49 residues affects its interaction with Atg24 and causes dominant-negative effects on proliferation. Formation of Cvt vesicles cannot complete when Leu378 and Ile382 in the AHL are simultaneously substituted for Glu, although this mutant protein still fractionates with vesicle membrane. Moreover, Atg20 and its partner protein, Atg24, need to be released from the PAS for reusing after PI3P is dephosphorylated by Ymr1. Taken together, these results suggest that the BAR domain of Atg20 regulates the Cvt pathway and endomembrane trafficking via multiple mechanisms, and Atg20 and Atg24 are recycled after the completion of Cvt vesicles formation.

並列關鍵字

autophagy ； the Cvt pathway ； Snc1 retrieval ； Atg20 ； Bin/Amphiphysin/Rvs (BAR) domain ； amphipathic helix ； Cvt vesicle formation

參考文獻

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