Biogenesis, maturation and fusion of autophagosomes

自噬在细胞的分解代谢和质量控制中起着至关重要的作用，它可以根据细胞的代谢需要进行调节。这在氮饥饿期间变得尤为明显，当细胞将部分胞浆及其细胞器隔离成新形成的自噬小体时。自噬小体从头开始形成，从小泡的融合开始，小泡通过脂质转移生长成双层膜结构，然后关闭，最后与液泡融合。在这个项目中，我们将重点放在这个过程的两个主要方面，使用酵母作为模式生物：(I)如何实现吞噬细胞的协调形成(通过解剖关键蛋白Atg2、Atg18和Atg9的相互作用)？这将在目标1中解决。(Ii)作为Rab7/Ypt7激活剂的脂磷酸酶Ymr1和Mon1-Ccz1鸟核苷酸交换因子(Global)如何控制自噬体成熟和膜融合的步骤？这将是目标2的重点。在目标1中，我们将分析脂类转移到生长的吞噬体中是如何调节和调节的。在我们之前关于Atg2-Atg18介导的脂质转移和依赖于Atg9的脂质扰乱的体外重建工作的基础上，我们将结合冷冻电子显微镜的结构方法与脂转移和扰乱的功能分析来了解这三种蛋白质的串扰和PI3P的作用。这将包括对Atg2与Atg2-Atg18复合体的比较分析，以及对Atg2-Atg9界面的详细剖析，以确定与脂质门控有关的关键残基。我们推测，Atg2结合控制了Atg9接受和扰乱脂类的能力，从而促进了脂类的持续和有效转移。我们将进一步询问，通过识别亚磷酸盐及其在控制蛋白质活性中的作用，激酶Atg1是如何控制这一过程的。所有相关的部位都将通过体内实验对各自的突变体进行分析，以验证体外的发现。在目标2中，我们将探讨不同的关键蛋白如何促进自噬体成熟和融合。我们分析的中心将是a)脂肪磷酸酶Ymr1对PI3P含量的控制，以及b)Mon1-Ccz1复合体的激活机制及其与Atg8的串扰，以修饰Ypt7自噬小体。对于这两个分项目，都建立了纯化蛋白质和检测方法来监测各自的配体。我们将特别确定Atg1激酶复合体在Ymr1和Mon1-Ccz1调控中的作用。任何来自磷蛋白质组筛选的控制其活性的点突变都将通过体内自噬试验进行验证。

Autophagy is of central importance in the catabolism and quality control of cells, which can be tuned to the metabolic needs of the cells. This becomes particularly obvious during nitrogen starvation, when cells sequester part of their cytosol and its organelles into newly formed autophagosomes. Autophagosome form de novo, starting with the fusion of small vesicles, which grow by lipid transfer into a double membrane structure, followed by closure and finally fusion with the vacuole. Within this project, we will focus on two main aspects of this process, using yeast as model organism: (i) How is the coordinated formation of the phagophore achieved (by dissecting the interplay of the key proteins Atg2, Atg18 and Atg9)? This will be addressed in Aim 1. (ii) How do the lipid phosphatase Ymr1 and the Mon1-Ccz1 guanine nucleotide exchange factor (GEF) as a Rab7/Ypt7 activator control steps in autophagosome maturation and membrane fusion? This will be the focus of Aim 2. In Aim 1, we will analyze how lipid transfer to the growing phagophore is mediated and regulated. Based on our previous work on the in vitro reconstitution of the Atg2-Atg18 mediated lipid transfer and the Atg9-dependent lipid scrambling, we will combine structural approaches by cryo-electron microscopy with functional analyses of lipid transfer and scrambling to understand the crosstalk of these three proteins and the role of PI3P. This will include the comparative analysis of Atg2 with the Atg2-Atg18 complex, and the detailed dissection of the Atg2-Atg9 interface to identify critical residues involved in lipid gating. We postulate that the Atg2 binding controls the ability of Atg9 to accept and scramble lipids, thus promoting a continuous and efficient transfer of lipids. We will further ask, how the kinase Atg1 controls this process by identifying phosphosites and their role in controlling the proteins’ activity. All relevant sites will be analyzed by in vivo assays with respective mutants to validate the in vitro findings. In Aim 2, we will ask how distinct key proteins promote autophagosome maturation and fusion. At the center of our analysis will be a) the control of the PI3P content by the lipid phosphatase Ymr1, and b) the mechanism of Mon1-Ccz1 complex activation and its crosstalk with Atg8 to decorate autophagosomes with Ypt7. For both subprojects, purified proteins and assays have been established to monitor the respective ligands. We will determine in particular the role of the Atg1 kinase complex in the control of Ymr1 and Mon1-Ccz1. Any point mutant, derived from a phosphoproteomic screen, controlling their activity will be validated by in vivo autophagy assays.