Autophagy initiation by the Atg1 complex

描述（由申请人提供）：自噬是细胞在饥饿期间存活和清除细胞中受损、有毒或过量的细胞器和内含物的保守途径。大量的细胞质、细胞器和其他自噬货物被吸收在称为自噬体的双膜囊泡内。自噬对细胞功能和人类健康至关重要，但自噬体生物发生的物理基础几乎完全未知。提出的研究将产生，在接近原子的细节，自噬体生物发生的最早阶段的机制。我们假设自噬体双膜起始的关键事件是在自噬体前结构（PAS）上含有膜蛋白Atg9的高曲率囊泡聚集。这一事件是由Atg1复合体策划的，该复合体在酵母中由Atg1、Atg13、Atg17、Atg29和Atg31组成。后三个包括Atg17-Atg31-Atg29亚复合物，这是第一个到达PAS的。我们确定了Atg17-Atg31-Atg29复合物的结构，揭示了一个显著的s形双新月，并提出了一个囊泡支架模型。我们还发现Atg1的c端早期自噬靶向/系聚（EAT）结构域是高膜曲率和系聚高曲率囊泡的有效传感器。这些见解使我们对启动自噬体生物发生的囊泡的支架和系缚进行了详细的假设。本项目的具体目的如下：1。我们将了解Atg1 EAT结构域是如何拴住高度弯曲的囊泡的。栓系将在体外进行试验，并与酵母细胞的功能相关联。栓系的结构基础将通过扫描诱变和氢-氘交换耦合质谱法确定。2. Atg1复合物的三维结构将由SAXS和EM确定，并通过扫描诱变绘制结构界面，以建立该复合物的伪原子模型。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PAS。该模型系统将用于探测饥饿期间Atg13去磷酸化是否会触发Atg1复合物的系索能力形式的组装。总之，这些目标将在机制和结构细节上充实自噬起始的主要早期事件。

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.