Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University, Budapest, Hungary.
Hungarian Academy of Sciences, Premium Postdoctoral Research Program, Budapest, Hungary.
PLoS Genet. 2018 Apr 25;14(4):e1007359. doi: 10.1371/journal.pgen.1007359. eCollection 2018 Apr.
The autophagosomal SNARE Syntaxin17 (Syx17) forms a complex with Snap29 and Vamp7/8 to promote autophagosome-lysosome fusion via multiple interactions with the tethering complex HOPS. Here we demonstrate that, unexpectedly, one more SNARE (Ykt6) is also required for autophagosome clearance in Drosophila. We find that loss of Ykt6 leads to large-scale accumulation of autophagosomes that are unable to fuse with lysosomes to form autolysosomes. Of note, loss of Syx5, the partner of Ykt6 in ER-Golgi trafficking does not prevent autolysosome formation, pointing to a more direct role of Ykt6 in fusion. Indeed, Ykt6 localizes to lysosomes and autolysosomes, and forms a SNARE complex with Syx17 and Snap29. Interestingly, Ykt6 can be outcompeted from this SNARE complex by Vamp7, and we demonstrate that overexpression of Vamp7 rescues the fusion defect of ykt6 loss of function cells. Finally, a point mutant form with an RQ amino acid change in the zero ionic layer of Ykt6 protein that is thought to be important for fusion-competent SNARE complex assembly retains normal autophagic activity and restores full viability in mutant animals, unlike palmitoylation or farnesylation site mutant Ykt6 forms. As Ykt6 and Vamp7 are both required for autophagosome-lysosome fusion and are mutually exclusive subunits in a Syx17-Snap29 complex, these data suggest that Vamp7 is directly involved in membrane fusion and Ykt6 acts as a non-conventional, regulatory SNARE in this process.
自噬体 SNARE 突触融合蛋白 17（Syx17）与 Snap29 和 Vamp7/8 形成复合物，通过与 tethering complex HOPS 的多重相互作用促进自噬体-溶酶体融合。在这里，我们出乎意料地证明，另一个 SNARE（Ykt6）对于果蝇中的自噬体清除也是必需的。我们发现 Ykt6 的缺失会导致自噬体的大规模积累，这些自噬体无法与溶酶体融合形成自溶酶体。值得注意的是，Syx5 的缺失，即 Ykt6 在 ER-Golgi 运输中的伴侣，不会阻止自溶酶体的形成，这表明 Ykt6 在融合中起着更直接的作用。事实上，Ykt6 定位于溶酶体和自溶酶体，并与 Syx17 和 Snap29 形成 SNARE 复合物。有趣的是，Ykt6 可以被 Vamp7 从这个 SNARE 复合物中竞争出来，我们证明 Vamp7 的过表达可以挽救 ykt6 功能丧失细胞的融合缺陷。最后，我们发现 Ykt6 蛋白的零离子层中的 RQ 氨基酸发生点突变形式（该突变形式被认为对于融合活性 SNARE 复合物的组装很重要）保留了正常的自噬活性，并在突变动物中恢复了完全的生存能力，而棕榈酰化或法尼基化位点突变的 Ykt6 形式则不然。由于 Ykt6 和 Vamp7 对于自噬体-溶酶体融合都是必需的，并且在 Syx17-Snap29 复合物中是相互排斥的亚基，这些数据表明 Vamp7 直接参与膜融合，而 Ykt6 在这个过程中充当非传统的调节 SNARE。
