阿斯加德古菌ESCRT剪切膜结构的分子机制研究

真核细胞内膜系统的起源机制一直是亟待解决的前沿科学问题。内体分选转运复合体（ESCRT）是真核细胞特有的在内膜系统中发挥膜剪切作用的复合体。现有研究表明，阿斯加德古菌中存在并表达ESCRT相关基因。申请人前期确证阿斯加德古菌ESCRT核心亚基Vps4、Vps2/24/46和Vps20/32/60在结构和功能方面类似真核生物同源蛋白。本项目进一步提出科学假设：阿斯加德古菌ESCRT对膜结构剪切作用的分子机制与真核生物相类似。为证明假设，项目将先利用等温滴定量热技术和GST pulldown等分析该古菌ESCRT各亚基组装模式；其次基于巨大单层囊泡系统分析其对膜结构的剪切作用；最后采用YeastFab Assembly等技术尝试将该古菌ESCRT整合至酿酒酵母，评估其在内膜系统中潜在生物学功能。本项目将探讨ESCRT由古菌向真核生物进化的可能性，为丰富真核生物及其内膜系统起源理论提供科学依据。

The emergence of endomembrane system is a key step in eukaryogenesis and has been a long-time dispute question. Most recently, homologues of Endosomal Sorting Complex Required for Transport (ESCRT) that has long been believed to be strictly conserved in eukaryotes were discovered in genomes of Asgard archaea, a newly proposed archaeal superphylum. It is thus assumed that eukaryotes originate from complex archaea, and this process gives rise to the endomembrane system. We have found that the core components of Asgard ESCRT, including Vps4, Vps2/24/46, and Vps20/32/60, have co-evolved eukaryote-like structural and functional features. These results further bring a question that whether there is a relevance of molecular mechanism and biological function between the Asgard archaeal ESCRT and its eukaryotic counterparts. In this study, assembly of the Asgard ESCRT is characterized by isothermal titration calorimetry and GST pulldown. Then, membrane-scission process of the machinery is reconstructed using giant unilamellar vesicles, fluorescence co-localization, and scanning electron microscopy. Finally, the Asgard ESCRT will be expressed in Saccharomyces cerevisiae to evaluate the functional similarity between the Asgard ESCRT and its eukaryotic counterparts. Taken together, this study focusses on the possibility that the eukaryotic ESCRT originally inherits from an archaeal ancestor, and offers important scientific evidence for eukaryogenesis and the evolutionary origin of the endomembrane system.