酵母Vps21模块蛋白通过Vps34和PI3P调控自噬前体闭合的机制研究

Autophagy is a conserved degradation process to maintain cellular homeostasis in eukaryotic cells. Autophagy dysfunction may result in abnormal growth and development and disease. Autophagy is mainly regulated by autophagy-related (Atg) proteins and its process includes phagophore formation, elongation, closure to form autophagosome, autophagosome fusion with lysosome, and finally degradation in lysosome. However, it is still inconclusive which factors regulate phagophore closure to form autophagosomes and what the underlying mechanism is. There are emerging evidence showing that Rab proteins which regulate vesicular transport (vesicle trafficking) also regulate autophagy. We recently reported that a yeast Rab5/Vps21 module (including Vps21, its upstream activator and downstream effectors) regulates autophagy because the absence of any Vps21 module protein results in accumulated autophagosomal structures outside the vacuole as a cluster. Our latest preliminary data indicate that these accumulated autophagosomal structures are unclosed phagophores. In this proposal, we are going to confirm the non-closure character of these autophagosomal structures in Vps21 module mutants with biochemical, cellular and genetics methods. We will further investigate the interactions between Vps21 module proteins and the PI3P-producing protein Vps34 and the PI3P-binding proteins Atg20, Atg24 by yeast two hybrid, bimolecular fluorescence complementary assay (BIFC) and co-immunoprecipitation, and determine the biological significance of the detected interactions. We propose that the Vps21 module protein regulates phagophore closure through its effect on localizations of Vps34 and PI3P at the endosome and the phagophore assembly site (PAS). The obtaining results will contribute greatly to our understanding of autophagy regulation mechanism.

细胞自噬（自噬）是真核细胞内降解物质、维持稳态的一种重要生理过程，自噬障碍可导致生长发育异常及病变。以自噬蛋白调控为主的自噬过程可分为自噬前体形成、延伸、闭合形成自噬体，以及自噬体与溶酶体融合及降解等阶段，但有关调控自噬前体闭合的因子及机制仍未有定论。近来发现调控囊泡运输的Rab蛋白也调控自噬，我们报道了酵母中的Rab5/Vps21模块蛋白（包含Vps21及其上游激活因子和下游效应蛋白）缺失后在液泡外堆积成簇的自噬体状膜结构，不能完成自噬过程。我们的预实验显示这些堆积的自噬体状膜结构为未闭合的自噬前体。本项目将使用生化、细胞、遗传等方法进一步确认Vps21模块蛋白缺失细胞中自噬前体未闭合，并探讨Vps21模块蛋白与PI3P的生成蛋白和结合蛋白之间的互作及意义，来解析Vps21模块蛋白通过影响Vps34和PI3P的定位调控自噬前体闭合的可能机制。研究结果将为自噬调控机理的研究做出重要贡献。

细胞自噬（自噬）是真核细胞内降解物质、维持稳态的一种重要生理过程，自噬障碍可导致生长发育异常及病变。以自噬蛋白调控为主的自噬过程可分为自噬前体形成、延伸、闭合形成自噬体，以及自噬体与溶酶体融合及降解等阶段，但有关调控自噬前体闭合的因子及机制仍未有定论。近年发现调控囊泡运输的Rab蛋白也调控自噬，我们报道了酵母中的Rab5/Vps21模块蛋白（包含Vps21及其上游激活因子和下游效应蛋白）缺失后在液泡外堆积成簇的自噬体状膜结构，不能完成自噬过程。我们使用生化、细胞、遗传等方法发现Vps21模块蛋白缺失细胞中自噬前体未闭合。紧接着我们从两个主要的方面探讨了自噬前体闭合的调控机制，首先发现ESCRT复合体在Vps21的调控下通过与Atg17的互作被招募到自噬前体上行使封口功能。其次，我们也发现Vps21缺失后减少Vps21模块蛋白与PI3P的生成蛋白和结合蛋白之间的互作，这可能是Vps21模块蛋白调控自噬前体闭合的另一机制。我们的研究结果为阐述自噬前体闭合的调控因子与机制做出了重要贡献。