ATG14可通过牵引膜及稳定tSNARE复合物加速SYX17-SNAP29-VAMP8驱动的哺乳动物细胞自噬体与溶酶体的膜融合。基因敲低另一膜牵引蛋白HOPS阻碍了该膜融合；且HOPS的组份之一Vps33作为SM蛋白家族的一员，可能具有组装哺乳动物自噬的SNARE复合物功能。据此我们推测 HOPS和ATG14协同促进自噬膜融合，并将在体外重组系统中验证。首先，我们将检验HOPS是否能够通过特异性桥连自噬体和溶酶体，组装SNARE复合物，以及保护该复合物抵抗NSF/αSNAP的水解以促进膜融合。进一步，我们将比较HOPS与ATG14单独及共同存在时的膜桥连效率，并检测ATG14能否协同HOPS促进SNARE复合物的组装和保护其抵抗水解，以阐明其协同作用机理。此外，我们也将对SNARE复合物的回收以及膜融合的启动途径进行探讨。综上，该研究将使我们更全面了解膜融合的调控机制，为药物研发奠定基础。

Our previous work revealed that ATG14 can promote mammalian autophagosome- lysosome membrane fusion driven by SYX17-SNAP29-VAMP8 complex, through membrane tethering and stabilizing SYX17-SNAP29 complex. Genetic knockdown of HOPS was also observed to inhibit the membrane fusion between autophagosomes and lysosomes in mammalian cells. Vps33, as one subunit of HOPS complex and a SM protein, might help assembling SYX17, SNAP29 and VAMP8 into a functional complex in mammalian autophagy. Here we hypothesize that HOPS and ATG14 synergistically promote the membrane fusion in mammalian autophagy. To test this hypothesis, we will reconstitute autophagosome and lysosome membrane system in vitro. We first aim to illuminate whether HOPS can promote the membrane fusion by specifically bridging autophagosomes and lysosomes, by assembling SYX17-SNAP29-VAMP8 complex, and by making this trans-SNARE complex to be resistant to NSF/αSNAP. We further aim to examine the potential synergistic effect between HOPS and ATG14. We will compare their membrane tethering capacity, individually and collectively. We will also test whether ATG14 can assist HOPS in assembling SNARE complex, as well as in protecting this complex from hydrolysis. In addition, we will investigate the recycling of SNARE complex and the trigging mechanism of autophagy membrane fusion. In summary, this study will help us understand the molecular mechanisms and guide us for future drug design.