探討Atg9磷酸化修飾之調控與細胞自噬的關聯

The regulation of Atg9 phosphorylation and its role in autophagy pathway

曾于庭(Yu-Ting Tseng)

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(2012年)

https://doi.org/10.6342/NTU.2012.10095

摘要

於真核生物細胞 (eukaryotic cells) 中，細胞自噬 (autophagy) 為一具有高度保守性的物質降解與循環路徑，此機制可用以維持細胞內部的恆定(homeostasis) 以及生理功能的正常運作。當細胞自噬被誘發時，細胞內會產生雙層膜狀構造，包裹住部分細胞質與其內的胞器或大分子物質，所形成的自噬體 (autophagosomes) 隨後會與液胞或溶酶體 (vacuoles/lysosomes) 癒合進行分解。於出芽酵母菌 (Saccharomyces cerevisiae) 中尚存在一特有的細胞自噬途徑──細胞質至液胞傳遞途徑 (cytoplasm-to-vacuole targeting pathway, Cvt pathway)，此一機制即使在環境養分充足的情況下仍會持續運行。在眾多種類的細胞自噬蛋白質中，Atg9 (autophagy-related gene 9) 為最先被發現的嵌膜蛋白，且被認為在細胞自噬及細胞質至液胞傳遞途徑的調控中扮演了重要的角色。在本篇研究中，我們發現當細胞自噬被誘發時，Atg9在順行運送 (anterograde transport) 至自噬體前驅構造 (pre-autophagosomal structure, PAS) 的過程中會伴隨著產生高度磷酸化 (phosphorylation) 修飾；而其逆行運送 (retrograde transport) 至細胞內的Atg9周邊儲源 (peripheral pools/reservoirs) 時則會導致部分磷酸化狀態的消失。

雖然目前對於Atg9周邊儲源在細胞中的位置仍無一致看法，但從本篇研究中發現，在Atg9的C端表現一特定的胺基酸序列RXR，使其分布局限於內質網 (endoplasmic reticulum, ER) 時，細胞質至液胞傳遞途徑與細胞自噬過程皆受阻礙，顯示Atg9需先由內質網離開後，才能從周邊儲源運送到自噬體前驅構造，否則將導致細胞質至液胞運輸小泡 (Cvt vesicles) 或自噬體無法正常形成。此外，由於Atg9為一磷酸化蛋白質 (phosphoprotein)，因此我們著手尋找可能與其作用的激酶 (kinases) 和磷酸酶 (phosphatases)。初步實驗結果顯示， PPH21和SNF1可能並未在細胞自噬與Atg9的磷酸化調控中扮演重要的角色；另外，雖然剔除CKA2後Atg9的磷酸化程度有下降的情形，但剔除此二種基因並未對細胞質至液胞傳遞途徑造成明顯抑制效果，且只有在剔除CKA2的細胞中發現細胞自噬有部分的缺陷；令人意外則是，剔除LCB5的細胞中無法偵測到Atg9的蛋白質，而此缺陷株的細胞質至液胞傳遞途徑與細胞自噬途徑皆產生明顯缺陷，顯示Lcb5蛋白可能為調控細胞自噬作用的核心分子，其機轉則仍有待後續研究的釐清。

關鍵字

Atg9 ； 出芽酵母菌 ； 細胞自噬 ； 細胞質至液胞傳遞途徑 ； 磷酸化

並列摘要

Autophagy is a self-degradation and recycling process that is essential for cellular homeostasis and cellular quality control. This mechanism is highly conserved in all eukaryotic cells. During autophagy, a portion of cytosolic components, including macromolecules and/or targeted organelles, are sequestered into the double-membrane autophagosomes, which then fuse with the vacuoles/lysosomes for degradation. In the budding yeast Saccharomyces cerevisiae, a selective type of autophagy named the cytoplasm-to-vacuole targeting (Cvt) pathway is constitutively active under growth conditions. Among all Atg (autophagy-related gene) proteins, Atg9 is the first identified transmembrane protein required for both the Cvt pathway and autophagy. In this study, we found that Atg9 is a phosphoprotein, which is hyper-phosphorylated upon autophagy induction. The phosphorylation modification of Atg9 correlates with its subcellular localization to the PAS (pre-autophagosomal structure), and its dephosphorylation is concomitant with the retrograde transport from PAS to its peripheral pools.

Additionally, we observed that Atg9 possessing the RXR-type ER-retention signals resulted in defects in the Cvt pathway and autophagy. While the organelle identify of the Atg9 peripheral pools is still unresolved with mitochondria, endoplasmic reticulum (ER), and endosomes all having been evoked, our result suggested that before reaching the PAS, Atg9 should be able to move beyond the ER heading to its peripheral pools. Moreover, we searched for possible phosphatase and kinase candidates of Atg9. Our preliminary results implied that Pph21 and Snf1 may not be involved in the regulation of autophagy and phosphorylation modification of Atg9. Although the cka2 null strain showed a reduction in Atg9 phosphorylation, the Cvt activities was not significantly affected in both deletion mutants, and only deletion of CKA2 led to partial defects in the bulk autophagy. Furthermore, to our surprise, Atg9 was not detectable in the lcb5 null mutant cells, and the Cvt pathway and autophagy were blocked in this deletion background. Taken together, our results show that Cka2 may be a positive regulator of autophagy via modulating the phosphorylation of Atg9, whereas the roles of Lcb5 in Atg9 phosphorylation and autophagy regulation await further elucidation.

並列關鍵字

Atg9 ； Autophagy ； Phosphorylation ； Saccharomyces cerevisiae ； The Cvt pathway

參考文獻

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