自噬通路上COPII囊泡融合相关SNARE复合物的系统鉴定与功能分析

The induction of autophagy by nutrient deprivation leads to a rapid increase in the formation of autophagosomes, double-membrane vesicles that sequester cytoplasmic materials and transport them to lysosome/vacuole for degradation. ER-derived COPII vesicles have been shown to be membrane source for autophagosome formation. However, little is known about mechanism of COPII fusion in autophagy pathway. SNARE proteins are the key components of the membrane fusion machinery. SNAREs that are localized in opposing membranes drive membrane fusion by using the free energy that is released during the formation of SNARE complex, a four-helix bundle. Previous studies have shown that SNAREs from the same exocytic SNARE complex (Sso1/2-Sec9-Snc1/2) play different roles in autophagy and that ER-localized SNARE Ufe1 is exported in specific COPII vesicles for autophagosome biogenesis. Our preliminary data show two SNAREs localized in COPII vesicles are required for autophagosome biogenesis and can form complex with other SNAREs involved in autophagy. These observations suggest that specific SNARE complex forms during autophagy and mediates COPII vesicles fusion for autophagosome biogenesis. To characterize the SNARE complex involved in COPII fusion in autophagy, we propose to perform genome-wide screening in yeast Saccharomyces cerevisiae to identify all the SNAREs required for autophagosome formation, characterize the possible SNARE complex components using in vitro binding and liposome fusion assays. Functional analysis of SNARE complex involved in COPII fusion in autophagy will be performed in both yeast and mammalian cells. Characterization and functional analysis of SNARE complex involved in COPII vesicle fusion in autophagy will elucidate the molecular mechanism of COPII fusion during autophagosome formation, and provide new insights into how cells use SNAREs and COPII vesicles to rearrange membranes in response to environmental cues.

自噬是将细胞组分包裹进双层膜结构的自噬体并运送至溶酶体降解的过程，对维持细胞稳态、应对环境刺激至关重要。COPII囊泡是自噬体的重要膜来源，但膜融合机制不详。作为介导膜融合的重要分子，位于两个待融合膜体上的SNARE通过配对形成复合物驱动融合。参与自噬的SNARE并不完全遵循已知配对规律，且自噬过程中有些SNARE发生位置转移，申请人前期研究表明COPII囊泡上存在参与自噬体形成的SNARE，且能与其他参与自噬的SNARE形成复合物，推测自噬通路上存在特定的SNARE复合物介导COPII囊泡融合。本研究将以酿酒酵母为主要研究模型，系统筛选自噬体形成相关SNARE，从中鉴定自噬通路上介导COPII囊泡融合的SNARE复合物，分析其膜融合功能，揭示其调控自噬体形成的作用机制，从分子-复合物-细胞等层面阐明自噬通路上COPII囊泡融合机制，为深入了解SNARE蛋白作用方式和自噬调控规律提供新线索

自噬是将细胞组分包裹进双层膜结构的自噬体并运送至溶酶体降解的过程，对维持细胞稳态和应对环境刺激至关重要。源自内质网的COPII囊泡是自噬体的膜来源，但其与自噬相关膜结构的融合机制不详。作为介导膜融合的重要分子，位于两个待融合膜体上的SNARE通过Qa-Qb-Qc-R配对形成复合物驱动融合。为了鉴定自噬通路上介导COPII融合的SNARE复合物，本研究以酿酒酵母为主要研究模型，系统筛选所有参与自噬体形成相关SNARE，并按照以下标准寻找可能的SNARE复合物排列组合：1）包含至少一个COPII囊泡上的SNARE，2）SNARE配比符合常规的Qa-Qb-Qc-R模式，3）有膜融合活性。得到下列两种可能性：Sed5-Bos1-Bet1-Sec22 复合物和 Sso1/2-Sec9-Sec22复合物。我们随后分析了候选SNARE蛋白与自噬通路上自噬相关Atg蛋白的互作，发现Bet1与Atg的互作频率高于其他候选SNARE，与多种Atg蛋白互作，且有自噬隔离膜上的定位，此外Bet1含有 LIR motif并以此与自噬膜结构标记物Atg8互作，提示Bet1位于自噬隔离膜，其互作SNARE Sed5-Bos1-Sec22 位于COPII囊泡介导膜融合。且Sed5-Bos1-Bet1-Sec22复合物在自噬膜融合过程中的作用在高等生物中保守，在哺乳动物细胞中敲低此复合物组分同样导致自噬早期膜结构异常。