未闭合自噬体与溶酶体融合的鉴定及意义

Autophagy is a degradation process to recycle and reuse materials in eukaryotes. It is important for maintaining cellular homeostasis and surviving unfavorable stress conditions. Currently it is generally acknowledged in the field of autophagy that phagophores expand to enclose degradable cargos to form closed autophagosomes and mature before they fuse with lysosomes/vacuoles. However, no one reported whether the unclosed autophagosomes (phagophores) during the normal autophagy process or the abundantly accumulated unclosed autophagosomes during the defective autophagy process can fuse with vacuoles to enter into them. The applicant's lab (we) found that the optical density (OD) of strains, the availability of vacuolar hydrolases and the different cells of the same strain all affected the degrees of accumulation of phagophore clusters next to the outer vacuolar membrane when we studied the regulatory proteins for phagophore closure. We proposed that the unclosed autophagosomes can fuse with vacuoles to enter into them. By deleting hydrolase genes PEP4 and PRB1 in vps21Δ strain and prolonging nitrogen starvation time, we detected that the unclosed autophagosomes entered vacuoles abundantly with fluorescence observation, transmission electron microscopy observation, protease protection assay and other methods. We will continue to validate these phenotypes and study the regulation mechanism and physiology relevance for unclosed autophagosomes entering vacuoles in this project. The results of this study will subvert the existing knowledge of only closed autophagosomes fusing with vacuoles. They will also promote the understanding of autophagy process and regulation mechanisms.

自噬是真核生物中一种物质降解并循环利用途径，对维持细胞稳态，帮助细胞渡过逆境等有重要意义。目前自噬领域内普遍认为自噬过程中自噬前体延伸后包裹待降解物形成成熟的闭合自噬体后才能与溶酶体/液泡融合，但无人报道正常自噬过程中那些未闭合自噬体(即自噬前体)和异常自噬过程中大量堆积的未闭合自噬体是否可以与液泡融合进入液泡内。申请者实验室在研究自噬前体闭合的调控蛋白时发现菌液的浓度，细胞中蛋白酶缺失等均会影响自噬前体簇在液泡膜外的堆积程度，且同样条件下同一菌株中有些细胞不产生自噬前体簇，从而提出未闭合自噬体可以与液泡融合进入液泡内的假设。通过敲除1-2个蛋白酶基因和延长氮饥饿时间，采用荧光显微镜观察、电镜观察、蛋白酶保护实验等方法，检测到Vps21等缺失菌株中未闭合自噬体可以大量进入液泡。本项目将确认这一表型，探讨这一现象的调控机制及生理意义。研究结果将颠覆现有理论并促进对自噬调控方式及机制的了解。

自噬是真核生物中一种物质降解并循环利用途径，对维持细胞稳态，帮助细胞渡过逆境等有重要意义。目前自噬领域内普遍认为自噬过程中自噬前体延伸后包裹待降解物形成成熟的闭合自噬体后才能与溶酶体/液泡融合，而未闭合自噬体不能与液泡融合进入液泡内。我们前期在酿酒酵母中研究自噬前体闭合的调控蛋白和分子机制过程中发现Vps21/Rab5或ESCRT复合体亚基缺失造成的液泡外堆积的未闭合自噬体随液泡水解酶的缺失而增加，从而提出未闭合自噬体可以与液泡融合进入液泡内的假设。通过敲除1-2个蛋白酶基因和延长氮饥饿时间，采用荧光显微镜观察、电镜观察、冷冻聚焦离子束切割和冷冻电子断层扫描、蛋白酶保护实验等方法，检测到Vps21或ESCRT复合体亚基缺失菌株中液泡外的未闭合自噬体可以大量进入液泡，成为未闭合的单层膜结构，在有蛋白酶时会被降解循环利用。且发现进入过程受到Vam3, Ypt7和一些Vtc蛋白的调控。未闭合自噬体进入液泡一定程度上弥补了自噬过程中断的缺陷，有利于这些二倍体酵母突变体产孢延续后代。本项目研究对真核生物中因自噬体不能闭合造成的相关自噬缺陷表型的改善具有指导意义。