Autophagosomal YKT6 is required for fusion with lysosomes independently of syntaxin 17

Abstract

Macroautophagy is an evolutionarily conserved catabolic mechanism that delivers intracellular constituents to lysosomes using autophagosomes. To achieve degradation, lysosomes must fuse with closed autophagosomes. We previously reported that the soluble -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein syntaxin (STX) 17 translocates to autophagosomes to mediate fusion with lysosomes. In this study, we report an additional mechanism. We found that autophagosome-lysosome fusion is retained to some extent even in knockout (KO) HeLa cells. By screening other human SNAREs, we identified YKT6 as a novel autophagosomal SNARE protein. Depletion of YKT6 inhibited autophagosome-lysosome fusion partially in wild-type and completely in KO cells, suggesting that YKT6 and STX17 are independently required for fusion. YKT6 formed a SNARE complex with SNAP29 and lysosomal STX7, both of which are required for autophagosomal fusion. Recruitment of YKT6 to autophagosomes depends on its N-terminal longin domain but not on the C-terminal palmitoylation and farnesylation that are essential for its Golgi localization. These findings suggest that two independent SNARE complexes mediate autophagosome-lysosome fusion.

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).…”

Curvature-sensitive trans-assembly of human Atg8-family proteins in autophagy-related membrane tethering

“…Furthermore, they strongly suggest that membrane-anchored Atg8 proteins can trigger only a reversible membrane tethering reaction by themselves but not mediate an irreversible hemifusion or fusion events when any other protein components are not present. Additional fusogenic proteins acting together with Atg8 proteins may include autophagy-related SNARE-family proteins that have been reported to be targeted to autophagic compartments and involved in autophagosome formation and maturation [22,[48][49][50]. However, it should be noted that, since membrane attachment of cytosolic Atg8 proteins via the conjugation to a nonlamellar-prone PE lipid is proposed to take place at locally-curved unstable lipid bilayers [45,46,51], we will not exclude the possibility that the intrinsic fusogenic potency of membrane-anchored Atg8 proteins directly facilitate membrane fusion events specifically at the highly-curved, fusion-prone membrane segments of autophagic compartments, including the rim of the cup-shaped phagophore membrane.…”

Hijacking intracellular membranes to feed autophagosomal growth

“…YKT6 presents multiple domains required for its intracellular localization and the N‐terminal longin domain has been found to be necessary for its localization to autophagosomes. At the autophagosome–lysosome interface, YKT6 interacts with SNAP29 and with the lysosomal SNARE STX7 .…”