Study of the post-translational modification and cycling of Atg9 in autophagy regulation

細胞自噬為細胞分解大分子物質的途徑之一，通常在細胞處於壓力的情況下會被誘發，且在真核生物演化上具有高度的保守性。當外界養分供應缺乏時，細胞內會產生雙層膜狀構造，將細胞質內的大分子物質和細胞質，甚至整個胞器包裹起來，形成自噬體，接著自噬體與液泡或是溶小體癒合進行分解，而分解所釋放出的胺基酸則會被用來合成在壓力情況下所需要的蛋白質。此外，在養分充足的情況下，酵母菌會進行專一性的細胞自噬稱為細胞質至液泡傳遞途徑，將液泡內水解酵素的前驅物運送至液泡進行切割與活化。tg9是在所有參與調控細胞自噬的必要蛋白質中，第一個被發現的嵌膜蛋白。在自噬體形成的過程裡，Atg9會在細胞自噬體前驅構造與細胞質周邊胞器間循環，因此Atg9被認為在自噬體形成過程中，對雙層膜成份的運輸扮演著關鍵的角色。此外，Atg9對於將其他參與調控細胞自噬有關的蛋白質誘導到自噬體前驅構造很重要，因此我們假設當Atg9到達自噬體前驅構造後會被進行某種修飾，以傳遞訊息使其他細胞自噬有關蛋白質能夠來到自噬體前驅構造。本論文研究發現，在養分缺乏的情況下，Atg9的胺基端會被磷酸化，且磷酸化的現象與Atg9在細胞內的移動有關—亦即Atg9是在自噬體前驅構造處被磷酸化，而去磷酸化過程則是與Atg9從自噬體前驅構造離開同時發生。此外，將可能被磷酸化的Serine19替換成無法被磷酸化的Alanine，則細胞自噬以及細胞質至液泡傳遞途徑會產生缺陷，細胞只能於形成數量較少以及直徑較小的自噬體；若將Serine19替換成帶負電的Aspartic acid以模擬磷酸化的狀態，細胞自噬活性則會顯著上升，表示Atg9的磷酸化現象在細胞自噬上扮演著重要的角色。然而，將Atg9胺基端的Serine11，Thronine16以及Serine19同時都替換成帶負電的胺基酸並沒有更進一步的加強細胞自噬的活性，反而產生些微的缺陷。我們認為長片段的負電胺基酸可能使得Atg9無法正常的在細胞間移動，因此細胞自噬活性並沒有如預期般上升。Autophagy is a degradative pathway that is induced under stress conditions such as starvation and is highly conserved in all eukaryotes. After induction, a portion of the bulk cytoplasm is non-selectively enwrapped into double-membrane vesicles, named autophagosomes, and finally the autophagosomes are delivered to vacuole for degradation. The amino acids released from the degradation of the cytosolic cargo are then used for synthesis of proteins that is vital for survival under stress conditions. In Saccharomyces cerevisiae, a selective autophagy, called the cytoplasm-to-vacuole targeting (Cvt) pathway, constitutively executes under vegetative condition, selectively delivering two resident vacuolar hydrolases aminopeptidase I (Ape1) and α-mannosidase (Ams1) to the vacuole.tg9 is the first identified integral membrane protein among the other autophagy-related (Atg) proteins and is an essential protein for both the Cvt pathway and autophagy. Therefore, Atg9 is considered to be the putative candidate for carrying the membrane source for de novo double-membrane formation. During vesicle formation, Atg9 is recruited to the PAS (pre-autophagosomal structure) to facilitate vesicle formation probably by delivering lipid components. Upon vesicle completion, Atg9 is retrieved form the completed autophagosomes.ere, we found that the N-terminus of Atg9 is hyper-phosphorylated under nitrogen starvation condition. In mutants that Atg9 cannot be recruited to the PAS, Atg9 is not hyper-phosphorylated when cells are starved, indicating that this phosphorylation occurs at the PAS. In addition, in mutants that the retrograde transport of Atg9 is blocked, Atg9 is even hyper-phosphorylated in vegetative condition and the modification of Atg9 phosphorylation statuses is not altered according to the nutrient availability, suggesting that the de-phosphorylation of Atg9 is concomitant with the retrograde transport of Atg9 from the PAS to peripheral structures. Substituting the possible phosphorylation site Ser19 with Alanine leads to partial Cvt pathway defect and reduction of autophagy activity, which is correlated with the formation of smaller and fewer autophagosomes. On the contrary, replacing the Ser19 with Aspartic acid results in significant higher autophagy activity. These results suggest that the phosphorylation of Atg9 at Ser19 plays a crucial role in autophagy regulation. However, Atg9 mutants with multiple Asp or Glu substitutions have partial defect instead of further enhance autophagy activity. We hypothesize that the hyper-negative charge of Atg9 might influence its normal cycling and therefore the autophagy activity is not increased as expected.Acknowledgements..............ibstract in Chinese.............iibstract..............................iiintroduction........................1aterials and methods.......8esults................................13iscussion...........................28eferences..........................34ables.................................41igures ...............................4