ATG9 scrambles lipids in concert with ATG2 lipid delivery to directly grow the autophagosome

项目摘要/摘要 自噬小体将细胞碎片包裹到负责运输的双膜小泡中 这些货物通过溶酶体进行降解，从而维持细胞内环境的稳定。自噬是如何 生长，特别是将脂质输送到生长结构的方法一直是 争执。我们实验室青睐的模式是将脂质从源膜直接输送到膨胀膜 通过早期作用的自噬相关蛋白ATG2吞噬噬菌体，我们将其定义为散装脂质 传送器。如果ATG2是通过直接的脂类运输来培养噬菌体的，那么肯定有脂类翻转 允许双层的内部小叶聚集的活动。由于翻转脂肪的性质 蛋白质，它们必须至少跨越膜一次，以促进脂质跨越。Atg9是唯一的 自噬所需的跨膜蛋白，其功能尚待确定。初步数据 我们的合作者的研究表明，ATG9能够在没有ATP存在的情况下在体外翻转脂质，使 这是一个潜在的扰乱。这些数据，报道的与ATG2的直接相互作用，以及ATG9 ATG2基因敲除后的积累导致了ATG9是种子上的一种脂质扰乱酶的假设 通过ATG2直接输送脂类促进吞噬体膜扩张的膜。这 该项目旨在提供必要的培训，以实现未来的职业生涯的独立研究。 此外，这个项目将增加对一个关键的细胞生物学过程的理解，并反过来 为未来的治疗研究奠定了基础。 该提案概述了两个目标-目标1：确定ATG9是否在体内起到扰乱酶的作用，以及 因此，在自噬体扩增过程中，它是否被嵌入到供体或受体的膜中；目的2： 评估自噬体膜和ATG9囊泡的脂质成分以追踪血管形成的来源 自噬小体形成所需的脂类。目标1将评估与以下相关的生物学变化 通过生物化学和显微技术的强大组合来操纵ATG9蛋白质。 自噬的中断将通过几个经典的自噬进程读数来评估，例如LC3 斑点形成和自噬因子转换(即p62)，并将允许有效地筛选ATG9 在提交给突变体进行体外扰乱酶活性评估之前。AIM 2将利用高分辨率 定量脂质组学和几个捕获自噬小体形态中间体的基因敲除细胞系 首次测定哺乳动物自噬体膜成分。的组成 自噬小体预计与源膜的组成大致相同。最后，要分开 自噬膜污染货物，苯乙烯马来酸(SMA)纳米盘将是 用来严格评估构成自噬小体的脂类。这项拟议的研究将有助于 对自噬形成机制的新见解，并回答了该领域长达数十年的问题。

Project Summary/Abstract The autophagosome encapsulates cellular debris into double-membrane vesicles responsible for trafficking this cargo to the lysosome for degradation, thereby maintaining cellular homeostasis. How the autophagosome grows and particularly the method by which lipid is delivered to the growing structure has been a key area of dispute. The model favored by our lab is direct delivery of lipid from a source membrane to the expanding phagophore by the early-acting autophagy-related protein ATG2 that we have defined as a bulk lipid transporter. If ATG2 is growing the phagophore through direct lipid transport, then there must be lipid flipping activity that allows for the population of the inner leaflet of the bilayer. Due to the nature of lipid-flipping proteins, they must span the membrane at least once to facilitate lipid crossover. ATG9 is the only transmembrane protein required for autophagy, and its function remains to be determined. Preliminary data from our collaborator shows that ATG9 is capable of flipping lipids in vitro without the presence of ATP, making it a potential scramblase. This data, the reported direct interaction with ATG2, and the fact that ATG9 accumulates with ATG2 genes knocked out led to the hypothesis that ATG9 is a lipid scramblase on the seed membrane that facilitates membrane expansion of the phagophore through direct lipid delivery by ATG2. This project is designed to provide the training necessary to achieve a future career in independent research. Further, this project will increase the understanding of a crucial cell biological process and in turn lay the foundation for future therapeutic lines of inquiry. This proposal outlines two aims – Aim 1: Determine whether ATG9 functions as a scramblase in vivo, and if so, whether it is embedded in the donor or acceptor membrane during autophagosome expansion; Aim 2: Evaluate the lipid composition of the autophagosomal membrane and ATG9 vesicles to trace the origin of the lipids required for autophagosome formation. Aim 1 will assess changes in the biology correlating to manipulation of the ATG9 protein by a powerful combination of biochemistry and microscopy techniques. Disruptions in autophagy will be evaluated by several classic readouts of autophagy progression such as LC3 puncta formation and autophagy factor turnover (i.e. p62), and will allow for efficient screening of ATG9 mutants before submitting them to in vitro scramblase activity assessment. Aim 2 will leverage high resolution quantitative lipidomics and several knockout cell lines that trap autophagosome morphological intermediates to determine the membrane composition of the mammalian autophagosome for the first time. The composition of the autophagosome is expected to approximate the composition of the source membrane. Finally, to separate the autophagosomal membrane from contaminating cargo, styrene maleic acid (SMA) nanodiscs will be employed to rigorously assess the lipids that comprise the autophagosome. This proposed study will contribute new insight into the mechanism of autophagosome formation and answer a decades-long question in the field.