篩選細胞自噬運送物機制之探討與Atg9之功能分析

Abstract

在細胞處於缺乏養分的環境時，細胞自噬（autophagy）的活性會上升，將細胞質中非必需的蛋白質和胞器運送至溶小體或濾泡中分解，所產生的基本組成單體用以維持細胞在惡劣環境中的生理運作。在酵母菌（Saccharomyces cerevisiae）研究系統中發現，當細胞處於養份充足的生長環境時，細胞自噬仍維持低度的活性，負責運送濾泡水解酶aminopeptidase 1前趨物（prApe1）及α- mannosidase 1（Ams1），此途徑特稱為細胞質至濾泡傳遞途徑（cytoplasm-to-vacuole targeting pathway, Cvt pathway）。 早期研究發現，prApe1在被運送至濾泡的過程中，會先被集中至PAS（pre-autophagosomal structure），在此處使prApe1被雙層膜構造包圍形成完整的運送小泡。而在運送至PAS之前，prApe1會先聚集形成一大型複合體，而後其運送過程的受體Atg19會與此複合體結合，再經由Atg19相繼地與Atg11及Atg8作用將prApe1送至PAS。然而，我們研究發現，Atg19與此兩分子間的交互作用關係對於prApe1的挑選具有加成效應；換言之，prApe1的挑選是經由Atg19分別與Atg11及Atg8結合而使此過程達到最大效益，而非經由接續性的作用方式進行。 另外，Atg9為一嵌膜蛋白，目前研究推論Atg9被徵召至PAS的過程中可能同時將運送小泡形成所需的膜送往此處。而我們研究發現，在Atg11協助將被運送物prApe1送往PAS的過程中，Atg11會透過與Atg9的直接交互作用關係將Atg9徵召至PAS，促進運送小泡的形成。 另一方面，Atg9會受磷酸化修飾，其磷酸化的程度會隨外在營養環境變化而改變：當細胞處於養分充足的環境時，Atg9主要以未磷酸化或低量磷酸化形態存在；當細胞受到飢餓刺激時，Atg9出現較高比例的高量磷酸化形態。近一步分析發現，Atg9 N端前60個氨基酸為可能接收磷酸根的區域，而其中第19個絲氨酸突變後，細胞自噬過程中形成的運送小泡數量明顯降低，同時伴隨著細胞自噬活性下降，顯示Atg9第19個絲氨酸與細胞自噬活性調控密切相關。

Autophagy is a catabolic membrane trafficking process conserved in all eukaryotic cells. During autophagic transport, cargos are incorporated into double-membrane vesicles and transported to the lysosomes/vacuole for degradation. In general, autophagy is induced in response to starvation stress for maintaining the cytosolic amino acid pool. In the budding yeast Saccharomyces cerevisiae, one type of selective autophagy, called the cytoplasm-to-vacuole targeting (Cvt) pathway, constitutively delivers at least two resident vacuolar hydrolases aminopeptidase Ι (Ape1) and α-mannosidase (Ams1). Precursor of Ape1 (prApe1) is transported by either pathways depending on the nutrient condition. In previous studies, prApe1 is found assembling into a higher order complex and associating with its transport receptor Atg19. Through the interaction between Atg19 and Atg11, the complex is recruited to the pre-autophagosomal structure (PAS). Once the complex arrives at the PAS, phosphatidylethanolamine (PE)-conjugated Atg8 binds to Atg19 to ensure incorporation of the complex into the forming Cvt vesicle. However, parts of prApe1 are still successfully transported into the vacuole via autophagy in starved atg11Δ cells. Here we report that prApe1 could not be targeted to PAS and consequently failed to be delivered into the vacuole in atg8Δ atg11Δ double knockout cells. Thus we propose that Atg19 mediates dual prApe1 sorting arms though independent, instead of sequential, interaction with Atg11 and Atg8. In addition, during/after the sorting process, Atg11 is involved in recruitment of Atg9 to the PAS for vesicle formation through direct physical interaction. Furthermore, in the absence of prApe1, some of autophagy proteins including Atg11 are co-localized at multiple sites next to the vacuole, suggesting that these spots are still functional for vesicle formation and Atg11 may not be specific for prApe1 transport but involved in general selective event during autophagy. Finally, we find that Atg9 is a phosphoprotein, which is hyper-phosphorylated under nitrogen starvation condition. A substitution mutation at the potential phosphorylation site Ser19 results in retarded Cvt pathway and bulk autophagy, along with the decrease of autophagosome generated under starvation, suggesting a close relation between Atg9 Ser19 and autophagy control.