Autophagy initiation by the Atg1 complex

描述（由申请人提供）：自噬是饥饿期间细胞存活以及从细胞中清除受损、有毒或过量细胞器和内含物的保守途径。大量的细胞溶质、细胞器和其他自噬物质被吸收在一个被称为自噬体的双膜囊泡中。自噬是细胞功能和人类健康的核心，但自噬体生物发生的物理基础几乎完全未知。这项拟议中的研究将以接近原子的细节产生自噬体生物发生最早阶段的机制。我们假设自噬体双膜启动的关键事件是含有膜蛋白Atg 9的高曲率囊泡聚集在自噬体前结构（PAS）处。该事件由Atg 1复合物协调，该复合物在酵母中由Atg 1，Atg 13，Atg 17，Atg 29和Atg 31组成。后三个包括Atg 17-Atg 31-Atg 29亚复合物，它是第一个到达PAS的。我们确定的结构的Atg 17-Atg 31-Atg 29复合物，揭示了一个显着的S形双新月，并提出了一个模型的囊泡支架。我们还发现Atg 1的C-末端早期自噬靶向/栓系（EAT）结构域是高膜曲率和栓系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小泡的支架和束缚提出了详细的假设。本项目的具体目标如下：1.我们将了解Atg 1 EAT结构域如何束缚高度弯曲的囊泡。将在体外测定栓系并将其与酵母细胞中的功能相关联。将使用扫描诱变和氢-氘交换耦合质谱法来确定拴系的结构基础。2.将通过SAXS和EM确定Atg 1复合物的三维结构，并通过扫描诱变绘制结构界面，以开发复合物的伪原子模型。3.在中心假设的严格检验中，将使用基于Atg 9脂肽的前体囊泡模型在体外重建早期PAS。该模型系统将被用来探测是否Atg 13去磷酸化饥饿期间触发的拴系能力形式的Atg 1复合物的组装。总之，这些目标将在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.