Non-canonical role of the SNARE protein Ykt6 in autophagosome-lysosome fusion

Abstract

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.

Mammalian Atg8 proteins regulate lysosome and autolysosome biogenesis through SNARE s

“…Recent studies have uncovered the yeast SNARE, Ykt6, as important in autophagosome-vacuole/lysosome fusion in yeast (Bas et al, 2018;Gao et al, 2018), and that mammalian Ykt6 ortholog (Matsui et al, 2018;Takats et al, 2018) plays a potentially dominant (over Stx17) role in mammalian autophagosome-lysosome fusion. Whereas the exact mechanism of Ykt6 in maturation has not been agreed upon, with differences in proposed models (Matsui et al, 2018;Takats et al, 2018), it is worth noting that Ykt6 is also part of one of the retrograde trafficking routes from endosomes to TGN that includes GOS-28/ GOSR1 (Tai et al, 2004), an mAtg8-binding SNARE as discussed above. Nevertheless, our data here suggest that Stx17 still plays a very important role in autophagosomal flux, revealed in the requirement for a STX16/STX17 double KO to block autophagic flux using the conventional and well-accepted LC3 flux assay (Klionsky et al, 2016), and reflected in autophagic degradation of a diverse panel of substrates: mitochondria, peroxisomes, M. tuberculosis, and ribosomes.…”

“…In the context of autophagy, SNAREs have been studied at different stages along the autophagy pathway (Nair et al, 2011;Itakura et al, 2012;Moreau et al, 2013;Kimura et al, 2017). Recent studies have indicated that additional SNAREs may be required or even be dominant in this process (Matsui et al, 2018;Takats et al, 2018). Recent studies have indicated that additional SNAREs may be required or even be dominant in this process (Matsui et al, 2018;Takats et al, 2018).…”

A conformational switch driven by phosphorylation regulates Ykt6 activity in macroautophagy

“…Ykt6 has been recently shown to be implicated in two cognate SNARE complexes in autophagosomal/lysosomal fusion (Matsui et al, 2018; Takats et al, 2018). One complex consists of Stx7 (Qa SNARE localized in the lysosome) and SNAP29 (Qbc SNARE) (Matsui et al, 2018).…”

“…In addition to its roles in the secretory and endocytic pathways, Ykt6 has recently been implicated in macroautophagy (hereafter referred to as autophagy) (Gao, Reggiori, & Ungermann, 2018) (Bas et al, 2018) (Takats et al, 2018) (Matsui et al, 2018). Autophagy is a highly conserved intracellular degradation pathway of long-lived proteins, misfolded proteins, and damaged organelles (Mizushima & Komatsu, 2011).…”

“…Key SNAREs involved in this function are Stx7, Stx17, SNAP29, and Vamp7/8. Although recent studies have implicated Ykt6 in autophagosome/lysosome fusion in mammalian cells (Takats et al, 2018) (Matsui et al, 2018), there are discrepancies between specific findings regarding the role Ykt6 plays relative to other SNAREs. To date, mechanisms underlying Ykt6 recruitment to the autophagosome/lysosome interface remain unresolved.…”