Autophagosome closure by the ESCRT machinery

项目总结/摘要 这个项目的目标是解决一个基本的知识差距，如何吞噬细胞关闭形成 双膜自噬体在大自噬（以下简称自噬）过程中， 吞噬细胞在细胞质周围伸长并密封以产生双膜自噬体 与溶酶体融合以降解货物随着吞噬细胞边缘变窄，细胞膜必须经历 核分裂分离内膜和外膜的过程类似于核内体分选 运输所需的复合物（ESCRT）介导的膜断裂。使用我们优雅的HaloTag-LC 3 自噬体完成测定，我们提供了ESCRT机制的首次实验证据， 哺乳动物吞噬细胞关闭，并确定ESCRT-I亚基VPS 37 A作为关键因素， 下游ESCRT向吞噬细胞的募集。值得注意的是，我们发现，N-末端推定的 VPS 37 A的泛素E2变体（PUEV）结构域是自噬体闭合所独特需要的，但 用于其他ESCRT介导的膜解离过程，包括内体受体分选 和胞质分裂。隔室特异性靶向因子启动四种ESCRT的顺序募集 复合物（ESCRT-I、-II、-III和VPS 4）与膜断裂位点结合。而吞噬细胞特异性 含有VPS 37 A的ESCRT-I复合物的靶向因子未知，我们的初步研究 显示VPS 37 A PUEV与含有阴离子脂质和脂质的高度弯曲的膜相互作用， 包装缺陷，这些都是吞噬细胞边缘的特征。我们假设PUEV选择性地 与高度弯曲的吞噬细胞膜相互作用，将ESCRT-1靶向吞噬细胞。而且我们的 初步研究表明，ESCRT向吞噬细胞的募集需要蛋白质泛素化， LC 3/GABARAP结合机制，使我们相信，膜相关的稳定性， ESCRT-I需要与吞噬细胞相关的泛素化货物的额外相互作用。我们在一个理想中 在以下具体目的中测试我们的假设：（1）确定VPS 37 A如何靶向ESCRT-1 在自噬体生物发生过程中，与吞噬细胞结合并指导下游ESCRT的组装;（2） 鉴定自噬过程中ESCRT-I的吞噬细胞特异性靶向因子。由于自噬参与了 许多生理和病理过程，这些研究将有深远的影响， 人类健康和疾病。

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.