Biogenesis, maturation and fusion of autophagosomes

自噬体的生物发生、成熟和融合

• 批准号: 448135520
• 负责人: Professor Dr. Christian Ungermann
• 金额: --
• 依托单位: Arbeitsgruppe Biochemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/448135520?language=en
• 关键词: Biogenesis; maturation; fusion; autophagosomes

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.

自噬在细胞的催化和质量控制中具有核心重要性，其可以根据细胞的代谢需要进行调整。这在氮饥饿期间变得特别明显，当细胞将其部分胞质溶胶及其细胞器隔离到新形成的自噬体中时。 自噬体从头形成，开始于小泡的融合，小泡通过脂质转移生长成双膜结构，随后闭合并最终与空泡融合。在这个项目中，我们将集中在这个过程的两个主要方面，使用酵母作为模式生物：（i）如何协调形成的吞噬细胞实现（通过解剖的关键蛋白质Atg 2，Atg 18和Atg 9的相互作用）？这将在目标1中讨论。(ii)脂质磷酸酶Ymr 1和Mon 1-Ccz 1鸟嘌呤核苷酸交换因子（GEF）作为Rab 7/Ypt 7激活剂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，我们将分析脂质转移到生长的吞噬细胞是如何介导和调节的。基于我们之前在体外重建的Atg 2-Atg 18介导的脂质转移和Atg 9依赖的脂质混乱的工作，我们将通过冷冻电子显微镜结合联合收割机结构方法与脂质转移和混乱的功能分析，以了解这三种蛋白质的串扰和PI 3 P的作用。这将包括Atg 2与Atg 2-Atg 18复合物的比较分析，以及Atg 2-Atg 9界面的详细解剖，以确定参与脂质门控的关键残基。我们推测，Atg 2结合控制的能力，Atg 9接受和争夺脂质，从而促进了连续和有效的转移脂质。我们将进一步询问激酶Atg 1如何通过识别磷酸化位点及其在控制蛋白质活性中的作用来控制这一过程。将通过体内试验对所有相关部位进行相应突变体分析，以验证体外结果。在目标2中，我们将询问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）脂质磷酸酶Ymr 1对PI 3 P含量的控制，以及B）Mon 1-Ccz 1复合物激活的机制及其与Atg 8的串扰以用Ypt 7装饰自噬体。对于这两个子项目，已经建立了纯化的蛋白质和检测方法来监测各自的配体。我们将确定特别是在控制Ymr 1和Mon 1-Ccz 1的Atg 1激酶复合物的作用。通过体内自噬测定来验证源自磷酸蛋白质组学筛选的控制其活性的任何点突变体。