Atg9磷酸化修飾對其細胞內運輸及細胞自噬之影響

中文摘要細胞自噬是細胞分解大分子物質的途徑之一，在真核生物中具有高度的保守性，可以維持細胞體內的恆定。當細胞處於壓力環境，特別是營養缺乏時，細胞自噬會被誘發，將非必要的蛋白質包裹起來，再經由與溶體或者是酵母菌中的液胞癒合進而分解，而釋出的胺基酸則可以用來供應合成細胞生存所需的蛋白質。在細胞自噬過程中所形成的雙層膜狀囊胞稱為自噬體，與其他膜狀運輸小泡不同的是，自噬體的雙層膜構造並非直接來自已經存在的膜，而是經由重新組合而成，但至今仍沒有直接證據證實單位膜的最初來源。Atg9是在目前已知參與調控細胞自噬的蛋白質裡唯一的嵌膜蛋白，對於自噬體的形成過程也是必須的，並且在自噬體形成的不同時期，Atg9會分別在自噬體前驅構造及細胞周邊胞器出現，構成一個運輸循環，因此被認為可能是把磷脂質帶到自噬體前驅構造的攜帶者。我們假設位在自噬體前驅構造的Atg9蛋白存在某種轉譯後修飾，這樣的修飾可以傳遞訊息，讓其他下游調控細胞自噬的蛋白質移動到自噬體前驅構造以完成自噬體的形成。本篇論文研究發現，在養份缺乏所引發細胞自噬的情形下，Atg9會經過高度磷酸化修飾，此段磷酸化的區域是位於Atg9的胺基端，對於Atg9在細胞內的運輸及細胞自噬的功能相當的重要。研究利用將胺基端置換方式將可能被磷酸化的絲胺酸和蘇胺酸換成不能被磷酸化的丙胺酸模擬無法被磷酸化修飾的Atg9，結果顯示，不能被磷酸化的突變的確會造成細胞自噬活性的缺陷，也會造成Atg9在細胞體內的循環受阻；另一方面，當絲胺酸和蘇胺酸被置換成帶負電的胺基酸以模擬磷酸化現象時，此Atg9突變蛋白竟然可以自行從自噬體前驅構造離開回到細胞周邊胞器，而不需要已知參與調控此過程的Atg1蛋白的作用。此外，我們利用免疫沈降法分析發現不同程度磷酸化的Atg9對於Atg11的親和力也有所不同。綜合以上，我們認為磷酸化修飾對於Atg9在細胞體內的運輸及對於細胞自噬功能的調節扮演著重要的角色。

關鍵字

細胞自噬

並列摘要

Abstract Autophagy is an evolutionally conserved degradation pathway through all eukaryotes, mediating cytosolic components turnover to maintain cellular homeostasis. It can be induced by stress signal, especially starvation condition, to make an adaptive response to environmental stimulus, by degrading cytoplasm, including entire organelles, in lysosome, or corresponding compartment in yeast, the vacuole, for necessary cell survival. Different form other secretory pathway, the formation of double-membrane vesicles, termed autophagosomes, is considered to be de novo instead of budding from a pre-existing membrane. However, the identity of the lipid membrane source in remains uncertain. Among all Atg (autophagy-related gene) proteins, Atg9 is the only integral membrane protein required for autophagosome formation. Moreover, while in the process of autophagy regulation, Atg9 cycles between peripheral sites and the PAS (pre-autophagosomal structure) in distinct steps. To be more specific, Atg9 is recruited to PAS during autophagosome formation and retrieved back to cytosolic area upon autophagosome completion. Hence, Atg9 is thought to be the candidate of membrane carrier. Here, we proposed that Atg9 may undergo some kind of modification to discriminate the PAS-localized and non-PAS-localized Atg9 in order to pass down the signal and employ different downstream proteins in definite situations. Indeed, we found that Atg9 is a phosphoprotein. Starvation treatment induces autophagy activity and causes hyper-phosphorylation of Atg9. Furthermore, the Atg9 phosphorylation sites are mapped to its N-terminus, which is important for its cellular localization and function in autophagy. Followed analyses suggested that the N-terminal alanine substitution Atg9 mutants impaired autophagy activity and blocked its retrograde transport from PAS. On the contrary, a phosphorylation-mimic Atg9 mutant with aspartate/glutamate substitution revealed no typical PAS-localized punctate structures in cells with deletion of ATG1, which is essential for recycling Atg9 from PAS back to the peripheral sites. Moreover, the co-immunoprecipitation assay demonstrated that the Atg9N1A exhibited stronger affinity to its interacting protein, Atg11, than wild type Atg9 and Atg9N1D. Together, we concluded that the phosphorylation modification of Atg9 plays a key role in its trafficking and autophagy regulation.

並列關鍵字

Atg9 ； autophagy

參考文獻

Andrei-Selmer, C., Knuppel, A., Satyanarayana, C., Heese, C., and Schu, P.V. (2001). A new class of mutants deficient in dodecamerization of aminopeptidase 1 and vacuolar transport. J Biol Chem 276, 11606-11614.

Baehrecke, E.H. (2005). Autophagy: dual roles in life and death? Nat Rev Mol Cell Biol 6, 505-510.

Bergamini, E., Cavallini, G., Donati, A., and Gori, Z. (2003). The anti-ageing effects of caloric restriction may involve stimulation of macroautophagy and lysosomal degradation, and can be intensified pharmacologically. Biomed Pharmacother 57, 203-208.

Bernales, S., McDonald, K.L., and Walter, P. (2006a). Autophagy counterbalances endoplasmic reticulum expansion during the unfolded protein response. PLoS Biol 4, e423.

Bernales, S., Papa, F.R., and Walter, P. (2006b). Intracellular signaling by the unfolded protein response. Annu Rev Cell Dev Biol 22, 487-508.

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Atg9磷酸化修飾對其細胞內運輸及細胞自噬之影響

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