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Title: Non-canonical role of the SNARE protein Ykt6 in autophagosome-lysosome fusion}
Article:
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.
Introduction:
Macroautophagy( hereafter autophagy) is a conserved lysosomal self-degrading and self-renewal pathway in eukaryotic cells. The fusion of autophagosomes with late endosomes/lysosomes is a critical step of autophagy. Fusion of eukaryotic membranes requires several functionally conserved factors including Rab family GTPases, tethering complexes and the appropriate complex of soluble N-methylmaleimide sensitive factor attachment protein receptor( SNARE) proteins. Rab GTPases define membrane identity and recruit the appropriate tethering factors. Tethers bridge the two membranes, and in several cases help the assembly of the SNARE complex, too[1, 2]. Most SNAREs are membrane anchored proteins, and all of them contain either one or two alpha helical SNARE motifs. This special coiled coil motif allows these proteins to form heterotetrameric SNARE complexes. Based on the biochemical properties of this motif, SNAREs are classified as Qa, Qb, Qc or R types. Qa, Qb and Qc SNAREs contain a glutamine while R SNAREs contain an arginine in the central( zero) layer of the assembled SNARE bundle. A fusion-competent quaternary SNARE complex contains one of each in the following orientation: Qabc SNARES are present in the first membrane and the complementary R-SNARE is found in the opposite membrane. The formation of a QabcR quaternary trans-SNARE complex promotes membrane fusion. After the fusion, SNAREs remain zippered in the same membrane( referred to as a cis-SNARE complex) until the Nsf-αSNAP complex untangles the bundle, followed by recycling of the monomeric SNAREs[3]. In metazoans, Rab7 and Rab2 are the most important small GTPases mediating autophagosome-lysosome fusion through their interaction with the HOPS heterohexameric tethering complex[4–7]. HOPS not only acts as a tether, it also interacts with the autophagosomal Qa SNARE Syntaxin 17( Syx17), and helps the assembly of the Syx17-Snap29-Vamp7/8( Drosophila/mammalian, respectively) Qa-Qbc-R SNARE complex[8–11]. Vamp7 together with Sec22 and Ykt6 belongs to the longin subfamily among R-SNAREs, as all of them have a ~150 aa longin N-terminal domain( LD)[12], which distinguishes these from the brevin subfamily that have shorter N-termini. Sec22 and Vamp7 act in the Golgi to ER transport pathway[13] and late endosomal-lysosomal fusion events[14, 15], respectively. However, the role of Ykt6 is less well characterized. In contrast to Vamp7 and Sec22, Ykt6 has no transmembrane domain: it associates to membranes through its double lipid anchor. The C terminus of mammalian Ykt6 contains a CCAIM motif in which the more C-terminal cysteine is constitutively farnesylated, while the other cysteine is reversibly palmitoylated. This reversible post-translational modification promotes a switch between the open and the closed conformations of the protein. Depalmitoylated Ykt6 forms a closed hairpin structure, in which the longin domain folds back to the SNARE motif. As the farnesyl group is hidden in this conformation, the closed form of Ykt6 is soluble, and it becomes membrane-associated in the open, palmitoylated form[16–22]. This cycling between the cytoplasm and membranes makes Ykt6 extremely mobile, as it can travel between different compartments independent of membrane trafficking routes. Due to this high mobility, Ykt6 has an unusually diverse function in various vesicular pathways. Its role was described in the early secretory pathway[23–25], it may also promote secretion[26, 27], and finally, yeast Ykt6 interacts with HOPS and plays an important but incompletely understood role in vacuolar fusion events[28–30]. During autophagy, yeast Ykt6 was suggested to promote phagophore assembly and autophagosome formation, rather than fusion[31]. The regulation of metazoan autophagosome-lysosome and yeast homotypic vacuole fusions show some similarities[32]. In yeast cells, fusion is mediated by HOPS and Ypt7/Rab7[33]. Although vacuolar SNAREs are not conserved( except for the Qb Vti1), all of these show structural and functional similarities to their metazoan analogs. Vam3 is a syntaxin-like Qa, Vam7 is a SNAP-like Qc[34] and finally Nyv1 is a Vamp7-like longin family R-SNARE with a transmembrane domain[35]. One could speculate that metazoan Ykt6 and Vamp7 may also coexist on the surface of lysosomes, and the role of metazoan Ykt6 is unclear. Here we show that Ykt6 is essential for autophagosomal clearance in Drosophila larval fat cells, and that Ykt6 is not functionally redundant with Vamp7. We demonstrate the competition between Ykt6 and Vamp7 for SNARE complex formation with Syx17 and SNAP29, and show that both lysosomal R SNAREs interact with HOPS through their longin domains. Finally, we demonstrate that the zero layer arginine is not essential for Ykt6 function. These findings suggest that Ykt6 acts as a non-conventional, regulatory SNARE in the course of autophagosome-lysosome fusion.
Discussion:
In this work, we demonstrated that the R SNARE Ykt6 is essential for autophagosome-lysosome fusion and it is present on lysosomes and autophagic vesicles in Drosophila. We also showed that Ykt6 can form a SNARE complex with Syx17 and Snap29, but this complex is less stable than the Syx17-Snap29-Vamp7 bundle. These findings, together with our observation that the zero layer mutant Ykt6 is fully functional unlike in yeast, raise the possibility that Ykt6 forms an only partially zippered SNARE complex, perhaps due to incompatibility between the C-terminal parts of Ykt6 and Syx17 SNARE motifs. Recently, stabilization of the C-terminal region of the assembled SNARE bundle was shown to be required for vesicle fusion[54]. Based on the differences between Vamp7 and Ykt6 in structure prediction and their relative binding affinity to Syx17 and Snap29, it seems unlikely that Syx17-Snap29-Ykt6 represents a classical, fusion-competent SNARE complex. The N-terminal part of Syx17 as well as the longin domains of Ykt6 and Vamp7 all bind to the HOPS central subunit Dor/Vps18 but not to Car/Vps33A( which may only bind SNARE domains), suggesting that these HOPS-SNARE interactions may stabilize and proofread the forming SNARE complexes, and that SNAREs present on different vesicles may promote tethering by recruiting HOPS. The cycling of Ykt6 between vesicles and the cytosol makes Ykt6 an excellent candidate as a key regulator of vesicle tethering and fusion. The requirement of two R SNARES for the same fusion event could also be explained by the possible redundancy between Vamp7 or Ykt6, but a full block of autophagosome clearance is seen in both Vamp7 and Ykt6 loss of function cells[8], and Vamp7 binds to Syx17 and Snap29 much stronger than Ykt6 does. In fact, an earlier study reported that Ykt6 dissociates from the vacuole fusion site before the actual fusion is completed[18], also suggesting that yeast Ykt6 is not functionally redundant with the other R SNARE, Nyv1. Membrane fusion requires the presence of numerous necessary factors( SNAREs, tethers, small GTPases, etc.) at the same time. As Ykt6 is highly mobile and can shuttle between vesicles and the cytosol, it may temporarily stabilize the Syx17-Snap29 Qabc SNARE complex bound to HOPS until the appropriate transmembrane R SNARE Vamp7 enters the fusion site. In line with this, we demonstrated that overexpression of Vamp7 restores autophagosome-lysosome fusion in Ykt6 loss-of-function cells. It is also worth noting that a cytosolic, soluble pool of human Syntaxin 17 has been identified thanks to glycine zipper formation between the two transmembrane domains[10], so even a fully formed, cytosolic Syx17-Snap29-Ykt6 SNARE complex might be identified in future biochemical and cell fractionation studies. Other multisubunit tethering complexes such as GARP, Dsl1 or COG also bind to various SNARE complexes, and the Dsl1 complex interacts with the NTDs of Sed5 and Sec20 SNARE proteins[47, 55–57]. Thus, SNARE complexes may have two distinct roles during membrane fusion: to facilitate docking by taking part in the recruitment of multisubunit tethers, and to mediate fusion by SNARE zippering. Based on our findings, we propose a possible scenario for autophagosome-lysosome fusion( Fig 6): first, Syx17( and together with it Snap29) is recruited to autophagosomal membranes. Second, a not fully zippered, “pre-fusion” SNARE complex is assembled with Ykt6, which is possibly bound to lysosomes. Third, HOPS is recruited with the help of multiple SNARE interactions, involving both the N-terminal regions and SNARE domains, to promote tethering. Finally, the appropriate R SNARE Vamp7 arrives to the fusion site, displaces Ykt6, and forms the fusion competent SNARE complex together with Syx17 and Snap29. It is also worth pointing out that Ykt6 is involved in multiple types of intracellular vesicle fusion events, so the fusion regulatory activity of Ykt6 is likely broader and not restricted to autophagosome-lysosome fusion. The facts that a zero ionic layer mutant form of Ykt6 is fully functional during autophagy and that R164Q mutant animals are completely viable suggest that forming a fusion-competent SNARE complex may not be the main function of Ykt6 during vesicle tethering and fusion in Drosophila.
SNARE proteins are critical executors of most vesicle fusion events in eukaryotic cells . 4 SNARE domains assemble into a bundle to promote fusion . We have previously shown that Syntaxin 17 , Snap29 ( contributing 2 SNARE domains ) and Vamp7 form the SNARE complex executing autophagosome-lysosome fusion in Drosophila . Surprisingly , one more SNARE protein ( Ykt6 ) is also required in vivo for autophagosome-lysosome fusion . We find that Ykt6 can form a less stable complex with Syntaxin 17 and Snap29 than Vamp7 , because Vamp7 outcompetes Ykt6 . Ykt6 , Vamp7 and Syntaxin 17 all bind to the tethering complex HOPS to promote vesicle fusion . Ykt6 likely plays a non-canonical role in autophagosome-lysosome fusion , because its mutant form ( which is thought to be unable to assemble into a fusion-competent SNARE complex ) still rescues the fusion defect of ykt6 mutant cells , and it restores viability in mutant animals .