酵母菌aminopeptidase 1的結構分析及其對細胞自噬囊泡形成所扮演之角色

在酵母菌中，細胞質至液泡傳遞途徑(cytoplasm-to-vacuole targeting pathway；Cvt pathway)這生物合成運輸方法會利用細胞自噬機關把前驅物aminopeptidase I (precursor aminopeptidase I；prApe1)聚合物隔絕進Cvt囊泡進而運送到液泡；在那它會被蛋白酶水解去除掉氨基末端45個氨基酸而形成其成熟形態Ape1。prApe1被視為扮演著Cvt囊泡組成中鷹架的重要角色，它可以利用其氨基末端的45個胺基酸propeptide來促成更高階之聚合物的形成以及細胞自噬受器的辦認。在本篇論文中展示了解析度為2.5埃的Ape1 X-光繞射晶體結構，揭露它的十二元體結構是由二聚體和三聚體子單元組成而成的四面體外觀。Propeptide具有濃度依賴性的聚合現象而且會形成一個穩定的四聚體。利用結構根據的突變實驗證明了儘管有propeptide的存在，但破壞了單元間的接觸面會防止十二元體的形成和液泡運輸。此外，把propeptide融合到多個會形成四級結構的蛋白上再去檢查它們的液泡輸入，發現propeptide在這些鷹架蛋白上的三度空間分佈對囊泡形成是重要的。本篇論文提供了一個機械性的啟示來更進一步了解選擇性細胞自噬中的Cvt或細胞自噬囊泡生合成的機制。

關鍵字

細胞自噬； X-光繞射；細胞質至液泡傳遞途徑；酵母菌；液泡

並列摘要

In Saccharomyces cerevisiae, a constitutive biosynthetic transport pathway, termed the cytoplasm-to-vacuole targeting (Cvt) pathway, encapsulates precursor aminopeptidase I (prApe1) dodecamers in the form of a giant complex into a Cvt vesicle using the autophagic machinery, sorting it into the vacuole (the yeast lysosome) where it is proteolytically processed into its mature form, Ape1, by removal of an amino-terminal 45-amino acid propeptide. prApe1 is thought to serve as a scaffolding cargo vital for the assembly of the Cvt vesicle by presenting the propeptide to mediate higher-ordered complex construction and autophagic receptor recognition. This dissertation presents the molecular architecture of Ape1 at 2.5 Å resolution and reveals its dodecameric architecture consisting of dimeric and trimeric units, which associate to form a large tetrahedron. The propeptide of prApe1 exhibits concentration-dependent oligomerization and forms a stable tetramer. Structure-based mutagenesis reveals that disruption of the inter-subunit interface prevents dodecameric assembly and vacuolar delivery in vivo despite the presence of the propeptide. Furthermore, by examining the vacuolar import of propeptide-fused exogenous protein assemblies with different quaternary structures, the 3-dimensional spatial distribution of propeptides presented by a scaffolding cargo is found to be essential for the assembly of the Cvt vesicle for vacuolar delivery. This dissertation provides the first mechanistic insight for understanding the autophagosomal biogenesis in selective macroautophagy.

並列關鍵字

Ape1 ； autophagy ； cytoplasm-to-vacuole targeting pathway ； dodecamer ； X-ray

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. The Journal of biological chemistry 276, 11606-11614.

Apostolovic, B., Danial, M., and Klok, H.A. (2010). Coiled coils: attractive protein folding motifs for the fabrication of self-assembled, responsive and bioactive materials. Chem Soc Rev 39, 3541-3575.

Araki, Y., Ku, W.C., Akioka, M., May, A.I., Hayashi, Y., Arisaka, F., Ishihama, Y., and Ohsumi, Y. (2013). Atg38 is required for autophagy-specific phosphatidylinositol 3-kinase complex integrity. The Journal of cell biology 203, 299-313.

Baba, M., Osumi, M., Scott, S.V., Klionsky, D.J., and Ohsumi, Y. (1997). Two distinct pathways for targeting proteins from the cytoplasm to the vacuole/lysosome. J Cell Biol 139, 1687-1695.

Bailey, S. (1994). The Ccp4 Suite - Programs for Protein Crystallography. Acta Crystallogr D 50, 760-763.

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酵母菌aminopeptidase 1的結構分析及其對細胞自噬囊泡形成所扮演之角色

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