酵母中的Ypt51与CORVET复合体互作对细胞自噬的影响及作用机理

Autophagy is a conserved process for degrading abnormal materials or organells in eukaryotic cells, and is required in multiple physiological processes, including growth, development, proliferation and stress resistance. It was reported that some Rab GTPases and their related regulators and effectors, whose main functions are in vesicle trafficking, are also involved in autophagy, but the mechanisms are still unclear. We recently found that Rab GTPase Ypt51, its guanine excahnge factor (GEF) Vps9, and its effectors Vps3 or Vps8 (specific-subunits of CORVET, class C core vacuole-endosome transport) are all involved in autophagy. Dysfunctioning one of these four proteins resulted in accumulation of autophagical protein Atg8 between the nucleus and the vacuole as banana-shaped structures, significantly different from the multiple Atg8 dots in the dysfunction of Ypt7, or its effector proteins Vps39 or Vps41 (specific-subunits of HOPS, homotypic vacuolar fusion and protein sorting). Based on our new finding of the functions of these four proteins in autophagy, we intent to explore the coordination of vesicle trafficking and autophagy between early endsome and late endosome. Fluorescence microscopy, electron microscopy, western blot and in vivo experiments would be used to examine: 1) the effect of either protein dysfucntion on autophagy; 2) suppression effect of Ypt51 on the defective autophagy of the dysfunction of other three proteins; 3) the direct interaction between these four proteins and autophagical proteins, such as Atg8, Atg9 and Ape1 etc.; 4) the binding activity on autophagosomes through the interaction between active Ypt51 and CORVET complex. These results will uncover the regulatory role of Ypt51 on autophagy, and provide mechanistic insight on how Rab small GTPases affect autophagy. This project will provide important theory base for mechanism study of autophagy.

细胞自噬是真核生物中降解非正常物质或细胞器的一种保守机制，参与多种重要的生理过程。有报道参与囊泡运输的Rab小G蛋白及相关调控因子也会影响细胞自噬，但具体机制不明确。我们最近发现酵母Rab小G蛋白Ypt51及其上游激活因子Vps9和下游效应物Vps3、Vps8（属于CORVET复合体专一性亚基）均参与了细胞自噬，缺失后细胞内都产生香蕉状结构。本项目在这些新发现的基础上，探讨囊泡运输与细胞自噬这两种生理过程在早期内体和后期内体之间如何偶联。本研究将采用活体荧光和电镜观察、免疫印迹检测及体外离体实验等方法探讨上述四个蛋白：缺失对细胞自噬的影响；过量表达Ypt51对其它三个蛋白缺失所产生自噬表型的恢复情况；与自噬蛋白的互作；激活的Ypt51通过与CORVET复合体互作结合自噬体的情况。本研究结果将揭示酵母中参与囊泡运输的Ypt51调控细胞自噬的机制，为细胞自噬调控机制研究提供重要理论基础。

本项目以酿酒酵母中参与囊泡运输的Rab小G蛋白及相关调控因子在细胞自噬中的可能作用为研究对象，主要研究了一系列蛋白（包括Ypt51/Vps21模块蛋白、Ypt31及TRAPPII复合体亚基、Ypt6等）缺失或突变对细胞自噬的影响，并构建这些蛋白在细胞自噬过程中的相互关系。发现这些Rab小G蛋白及相关调控因子在细胞自噬中具有非常重要且不同的作用。得到如下重要结果：1）以Ypt51/Vps21为中心的模块蛋白（包括激活因子、小G蛋白、效应因子）缺失导致自噬过程缺陷，自噬体在液泡外堆积，无法进入液泡中降解，这些自噬缺陷表型的产生可能与这些蛋白缺失导致PI3K复合体亚基错误定位，从而导致自噬体无法封口，不能完成正常的自噬过程有关；2）不同的TRAPP复合体亚基通过不同的Rab小G蛋白调控细胞自噬；3）Ypt1可以互补Ypt6及其激活蛋白缺失所造成的自噬缺陷。这些结果说明，由Rab小G蛋白及相关调控因子所调控的正常完整的囊泡运输过程是保证细胞自噬正常进行的前提。基于本项目已在知名期刊Molecular biology of the Cell等杂志上发表SCI论文4篇，另有至少3篇SCI论文待发表。这些研究成果对阐释参与囊泡运输的Rab小G蛋白及相关调控因子在细胞自噬中的作用提供了坚实的实验数据，对推动细胞自噬调控机制的研究做出了一定的贡献。