探討Atg24之功能與結構在細胞自噬上所扮演的角色

Abstract

細胞自噬(Autophagy)屬於一種分解機制，在真核生物中具有高度的演化保守性。它與許多細胞生理反應有關，且受到細胞自噬相關蛋白(Atg)的精密調控。在細胞自噬進行的過程中，具有雙層膜構造的自噬小體(Autophagosome)會將細胞質中的目標物包裹起來，並且與液胞(Vacuole)融合，最後被水解酶所分解。細胞自噬除了可以非選擇性地包裹目標物外，也可以專一性地包裹多餘的胞器進行降解作用。在出芽酵母菌中(Saccharomyces cerevisiae)，養分的缺乏會誘發非選擇性細胞自噬的進行；然而酵母菌特有的選擇性細胞自噬：細胞質至液胞傳遞(Cytoplasm-to-vacuole targeting, Cvt)途徑，即使是在養分充足的環境下，仍可以持續地進行。雖然此兩種細胞自噬具有相似的調控機制，但在調控過程中仍然有特殊的蛋白只專一性地參與在其中一種細胞自噬之中。Atg24是一種可以和磷脂醯肌醇3-磷酸(PI3P)結合的蛋白，它僅調控選擇性細胞自噬，而不參與在非選擇性細胞自噬。Atg24除了具有可和PI3P結合的模組(domain)之外，也同時兼具兩個螺旋(coiled-coil)模組和雙性螺旋(Amphipathic helix)複合的BAR模組。其中BAR模組具有可以感受生物膜曲度的功能，因此可以藉由幫助磷脂質在膜間的傳遞以及囊泡之間的融合來調控膜系統之間的運作。在本篇研究中，主要在探討Atg24的功能與結構對於細胞自噬的調控機制。首先，我們發現Atg24透過其羰基端與Atg20進行交互作用。其次，Atg24的BAR模組除了為細胞質至液胞傳遞途徑所必需之外，也影響內膜系統的傳遞和液胞蛋白的運送。除此之外，我們也證實Atg24的BAR模組對於其本身與膜之間的結合很重要。總結來說，Atg24參與在PI3P訊息傳遞之後的調控步驟，並且透過BAR模組的特性來調控細胞自噬的進行。

Autophagy, a degradation pathway, is highly conserved in all eukaryotes. It is associated with many physiological processes and elaborately regulated by various autophagy-related (Atg) proteins. In autophagy, a double-membrane vesicle named the autophagosome engulfs cytoplasmic cargoes and fuses with the lysosome/vacuole, which results in the breakdown process through the actions of vacuolar hydrolases. Generally, autophagy is considered a non-specific process, but there are selective autophagy pathways functioning in the degradation of excess organelles. In Saccharomyces cerevisiae, nonselective autophagy is highly induced under starvation environment, but a particular type of selective autophagy, the cytoplasm-to-vacuole targeting (Cvt) pathway, is constitutively active and even operates in nutrient-rich condition. Although these two pathways share the same core machinery, the specific regulators for each pathway are also required. Atg24, a PI3P-binding protein, specifically regulates the Cvt pathway, but is dispensable for the bulk autophagy. Furthermore, it is involved in the endosome trafficking pathway. Except the PI3P-binding domain, domain structure analysis of Atg24 identifies two coiled-coil domains and a BAR domain associated with amphipathic helix. The BAR domain is considered a membrane-curvature sensor. Therefore, BAR-domain containing proteins regulate membrane trafficking through facilitating lipid transfer between membranes and tethering of vesicles. In this study, I functionally characterized Atg24 in autophagy regulation. First, I found that Atg24 interacts with Atg20 through a region between the two coiled-coil domains at the C-terminus. This interaction is critical to the Cvt pathway, because Atg24 and Atg20 promote Cvt vesicle formation in a mutually dependent manner. Next, I investigated that the BAR domain of Atg24 is required for endosome trafficking, vacuolar protein sorting, and the Cvt pathway. Furthermore, I suspected that the BAR domain is essential for Atg24 binding to lipid membrane. In conclusion, I proposed that Atg24 functions after PI3P signaling in the progress of the Cvt pathway and regulates selective autophagy through the characteristic BAR domain.