Autophagy initiation by the Atg1 complex

描述(由申请人提供)：自噬是一种保守的途径，用于在饥饿期间细胞存活，并清除细胞中受损的、有毒的或多余的细胞器和内涵体。大量的细胞液、细胞器和其他自噬货物被称为自噬小体的双层膜小泡吸收。自噬是细胞功能和人类健康的核心，然而自噬生物发生的物理基础几乎是完全未知的。这项拟议的研究将以接近原子的细节，为自噬生物发生的最早阶段提供机制。我们推测，自噬小体双膜启动的关键事件是在自噬前结构(PAS)处包含膜蛋白Atg9的高曲率小泡的聚集。这一事件由Atg1复合体策划，在酵母中，Atg1复合体由Atg1、Atg13、Atg17、Atg29和Atg31组成。后三个组成了Atg17-Atg31-Atg29亚复合体，它是第一个到达PAS的。我们测定了Atg17-Atg31-Atg29复合体的结构，发现了一个显著的S形状的双新月，并提出了一种囊泡支架的模型。我们还发现，ATg1的C末端早期自噬靶向/拴系(EAT)结构域是高膜曲率和拴系高曲率小泡的有效传感器。这些见解使我们对启动自噬小体生物发生的小泡的支架和拴系提出了一个详细的假说。本项目的具体目标如下：1.了解ATG1 EAT结构域是如何拴住高度弯曲的囊泡的。拴系将在体外进行测试，并与酵母细胞的功能相关。系链的结构基础将通过扫描诱变和氢-重离子交换与质谱学相结合来确定。2.用SAXS和EM确定Atg1复合体的三维结构，并通过扫描诱变绘制结构界面图，建立复合体的伪原子模型。3.在对中心假设的严格检验中，将使用基于ATg9脂肽的前体囊泡模型在体外重建早期的PAS。该模型系统将被用来探索饥饿期间Atg13的去磷酸化是否触发了一种具有拴系能力的Atg1复合体的组装。总而言之，这些目标将从机制和结构细节上充实自噬启动的主要早期事件。

DESCRIPTION (provided by applicant): Autophagy is a conserved pathway for cell survival during starvation and for clearance of damaged, toxic, or excess organelles and inclusions from the cell. Bulk cytosol, organelles, and other autophagic cargo are taken up within a double membrane vesicle known as the autophagosome. Autophagy is central to cell function and human health, yet the physical basis of autophagosome biogenesis is almost completely unknown. The proposed research will yield, in near-atomistic detail, the mechanism for the earliest stage in autophagosome biogenesis. We hypothesize that the key event in the initiation of the double membrane of the autophagosome is the clustering of high curvature vesicles containing the membrane protein Atg9 at the preautophagosomal structure (PAS). This event is orchestrated by the Atg1 complex, which in yeast consists of Atg1, Atg13, Atg17, Atg29, and Atg31. The latter three comprise the Atg17-Atg31-Atg29 subcomplex, which is the first to arrive at the PAS. We determined the structure of the Atg17-Atg31-Atg29 complex, revealing a remarkable S-shaped double crescent and suggesting a model for vesicle scaffolding. We also discovered that the C-terminal early autophagy targeting/tethering (EAT) domain of Atg1 is a potent sensor for high membrane curvature and tethers high curvature vesicles. These insights led us to a detailed hypothesis for the scaffolding and tethering of the vesicles that initiate autophagosome biogenesis. The specific aims of this project are as follows: 1. we will understand how the Atg1 EAT domain tethers highly curved vesicles. Tethering will be assayed in vitro and correlated with function in yeast cells. The structural basis for tethering will be determined using scanning mutagenesis and hydrogen-deuterium exchange coupled to mass spectrometry. 2. The three dimensional structure of the Atg1 complex will be determined by SAXS and EM, and structural interfaces mapped by scanning mutagenesis to develop a pseudo-atomic model for the complex. 3. In a stringent test of the central hypothesis, an Atg9 lipopeptide based model for precursor vesicles will be used to reconstitute the early PAS in vitro. The model system will be used to probe whether Atg13 dephosphorylation during starvation triggers the assembly of a tethering-competent form of the Atg1 complex. Together, these aims will flesh out the main early events of autophagy initiation in mechanistic and structural detail.