Autophagosome closure by the ESCRT machinery

项目摘要/摘要 这个项目的目标是解决关于吞噬细胞如何闭合形成的知识上的一个根本差距。 双膜自噬小体。在巨型自噬期间(下称自噬)，呈新月形 吞噬体在细胞质周围伸长并封闭以产生双膜自噬小体 与溶酶体融合以促进货物降解。当噬菌体边缘变窄时，膜必须经历 在类似于内体分选的过程中分裂分离内膜和外膜 转运所需的复合体(ESCRT)介导的膜断裂。使用我们优雅的HaloTag-LC3 自噬完成分析，我们为ESCRT机制提供了第一个实验证据。 并确定ESCRT-I亚单位VPS37A是导致ESCRT-I亚基关闭的关键因素 将下游的ESCRT重新招募到噬菌体。值得注意的是，我们发现N末端推定 VPS37A的泛素E2变异体(PUEV)结构域是自噬小体关闭所必需的，但 对于ESCRT介导的其他膜脱落过程，包括内小体受体分选，是必不可少的 和胞质分裂。隔室特异性靶向因子启动了四种ESCRT的顺序招募 复合体(ESCRT-I、-II、-III和Vps4)到膜断裂部位。而吞噬动物特有的 我们的初步研究表明，含有VPS37A的ESCRT-I复合体的靶向因子尚不清楚 发现VPS37A PUEV与含有阴离子脂类和脂类的高度弯曲的膜相互作用 包装缺陷，这都是噬菌体边缘的特征。我们假设PUEV有选择地 与高度弯曲的吞噬体膜相互作用，将ESCRT-I靶向噬菌体。此外，我们的 初步工作表明，ESCRT在吞噬细胞中的募集需要蛋白质泛素化，并且 LC3/GABARAP接合机制，使我们相信稳定的膜缔合 ESCRT-I需要与与噬菌体相关的泛素化货物进行额外的相互作用。我们正处在一个理想中 在以下特定目标中测试我们的假设：(1)确定VPS37A如何靶向ESCRT-I 在自噬小体生物发生过程中吞噬并指导下游ESCRT的组装；(2) 确定自噬过程中ESCRT-I的噬菌体特异性靶向因子。因为自噬参与了 众多的生理和病理过程，这些研究将对 人类的健康和疾病。

Project Summary/Abstract The goal of this project is to address a fundamental gap in knowledge on how phagophores are closed to form double membrane autophagosomes. During macroautophagy (hereafter autophagy), crescent-shaped phagophores elongate around cytoplasmic material and seal to generate double-membrane autophagosomes that fuse with lysosomes for cargo degradation. As the phagophore rim narrows, the membranes must undergo fission to separate the inner and outer membranes in a process that bears resemblance to endosomal sorting complexes required for transport (ESCRT)-mediated membrane scission. Using our elegant HaloTag-LC3 autophagosome completion assay, we provided the first experimental evidence for the ESCRT machinery in mammalian phagophore closure and identified the ESCRT-I subunit VPS37A as critical factor for the recruitment of downstream ESCRTs to the phagophore. Notably, we found that the N-terminal putative ubiquitin E2 variant (PUEV) domain of VPS37A is uniquely required for autophagosome closure but is dispensable for other ESCRT-mediated membrane abscission processes, including endosome receptor sorting and cytokinesis. Compartment-specific targeting factors initiate the sequential recruitment of the four ESCRT complexes (ESCRT-I, -II, -III and VPS4) to the membrane scission site. While the phagophore-specific targeting factors for the VPS37A-containing ESCRT-I complex are unknown, our preliminary study has revealed that VPS37A PUEV interacts with highly curved membranes containing anionic lipids and lipid packing defects, which are all features of the phagophore rim. We hypothesize that the PUEV selectively interacts with highly curved phagophore membranes to target ESCRT-I to the phagophore. Furthermore, our preliminary work has revealed that ESCRT recruitment to phagophores requires protein ubiquitylation and the LC3/GABARAP conjugation machinery, leading us to believe that the stabilization of membrane-associated ESCRT-I requires additional interactions with phagophore-associated ubiquitylated cargo. We are in an ideal position to test our hypotheses in the following Specific Aims: (1) to determine how VPS37A targets ESCRT-I to phagophores and directs the assembly of downstream ESCRTs during autophagosome biogenesis; (2) to identify phagophore-specific targeting factors for ESCRT-I during autophagy. As autophagy is involved in numerous physiological and pathological processes, these studies will have far-reaching implications for human health and disease.