InteractiveFly: GeneBrief

YKT6 v-SNARE: Biological Overview | References

In the Drosophila larval salivary gland, developmentally programmed fusions between lysosomes and secretory granules (SGs) and their subsequent acidification promote the maturation of SGs that are secreted shortly before puparium formation. Subsequently, ongoing fusions between non-secreted SGs and lysosomes give rise to degradative crinosomes, where the superfluous secretory material is degraded. Lysosomal fusions control both the quality and quantity of SGs, however, its molecular mechanism is incompletely characterized. This study identified the R-SNARE Ykt6 as a novel regulator of crinosome formation, but not the acidification of maturing SGs. WYkt6 localizes to Lamp1+ carrier vesicles, and forms a SNARE complex with Syntaxin 13 and Snap29 to mediate fusion with SGs. These Lamp1 carriers represent a distinct vesicle population that are functionally different from canonical Arl8+, Cathepsin L+ lysosomes, which also fuse with maturing SGs but are controlled by another SNARE complex composed of Syntaxin 13, Snap29 and Vamp7. Ykt6- and Vamp7-mediated vesicle fusions also determine the fate of SGs, as loss of either of these SNAREs prevents crinosomes from acquiring endosomal PI3P. These results highlight that fusion events between SGs and different lysosome-related vesicle populations are critical for fine regulation of the maturation and crinophagic degradation of SGs (Szenci, 2024).

Professional secretory cells produce large amounts of secretory material (hormones, neuropeptides, digestive enzymes, mucin, etc.) and store them in secretory granules (SGs) until a secretagogue elicits their bulk exocytosis. These cells usually produce more secretory material than is released by exocytosis to provide a sufficient pool of available SGs. Secretory cells continuously turn over the excess SGs by crinophagy, a specialized form of autophagy to maintain a constant releasable pool of SGs. Following this route, abnormal or obsolete SGs may also be subject to crinophagic degradation. In addition to degradative crinophagy, SG-lysosome fusions may also contribute to the complex maturation process of SGs and thereby determine their controlled release by exocytosis. During crinophagy, SGs directly fuse with lysosomes that gives rise to degradative crinosomes (Szenci, 2024).

Easy genetic manipulation and highly conserved molecular mechanisms make Drosophila a powerful in-vivo model for deciphering the molecular regulation of the regulated secretory pathway and crinophagy. Salivary gland cells produce and secrete high amounts of Sgs (Salivary gland secretion)/glue proteins in response to peaks of the molting hormone ecdysone. The released glue is then expelled from the lumen to anchor the metamorphosing prepupae to solid surfaces. The nascent glue SGs emanate from the TGN1, increase in size by homotypic fusions, and then undergo a complex maturation process during which SGs fuse with lysosomes. This promotes the acidification and profound reorganization of the inner content of SGs preparing them for secretion. Excess or abnormal glue can be also degraded by crinophagy, through fusion of non-secreted SGs and lysosomes. Taken together, crosstalk and fusion between SGs and the endolysosomal compartment is critical both for SG maturation and crinosome formation, however, the molecular mechanism of these processes is still incompletely understood (Szenci, 2024).

By enabling direct fusion between SGs and lysosomes, crinophagy differs mechanistically from the canonical main autophagic pathway, which mediates the degradation of cytosolic material through autophagosome formation and their subsequent fusion with lysosomes. Accordingly, genes that are required for autophagosome formation proved dispensable to crinophagy, while SG-lysosome fusion itself relies on a similar molecular machinery acting in fusions between autophagosomes and lysosomes The machinery mediating autophagosome-lysosome fusion is well characterized both in Drosophila and humans by now. Critical components include Rab2, Rab7, and Arl8 small GTPases that also contribute to defining membrane identity, homotypic fusion and vacuole protein sorting (HOPS) tethering complex, and a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex (SNAREpin) that executes the fusion. Based on biochemical properties, functional SNAREpins assemble from three Q-(Qabc) and one R-SNARE domains. The first discovered SNAREpin that mediates autophagosome-lysosome fusion is composed of Syntaxin 17, Snap29 and Vamp7/8. Recently another R-SNARE: Ykt6 was also discovered to also have a role in the process, either as an R-SNARE potentially substituting for Vamp7 or interacting with Syntaxin 7 and Snap29 to form an alternative SNAREpin3. Interestingly, Drosophila crinophagic fusion of glue SGs and lysosomes depends on highly similar machinery, composed of Rab2, Rab7, Arl8, HOPS and a Syntaxin 13, Snap29 and Vamp7 SNAREpin3. The similarity of the molecular machinery regulating these lysosomal fusions raised the possibility that Ykt6 may also regulate SG-lysosome fusions and crinophagy (Szenci, 2024).

Ykt6 is a highly conserved R-SNARE that consists of an N-terminal longin domain (LD), an R-SNARE domain, and a conserved C-terminal lipidation motif with the amino acid sequence CCAIM. The latter is critical for membrane association because Ykt6, unlike other R-SNAREs, lacks a canonical transmembrane domain. Moreover, the lipid anchors can hide reversibly in the hydrophobic groove of the protein, which enables Ykt6 to leave membranes and form a cytosolic pool. This way, it can be rapidly incorporated into various intracellular membranes on demand and form a complex with compartment-specific Q-SNAREs to promote vesicle fusion. Membrane-associated Ykt6 regulates the anterograde ER to Golgi, the intra-Golgi4, retrograde directed Golgi to ER, and endosome to TGN transports, and the release of constitutive secretory carriers or exosomes along the secretory pathway. In addition, it also promotes biosynthetic transport to the yeast vacuole and lysosomes in animal cells, endosomal recycling, and macroautophagic degradation. However, the role of Ykt6 in SG-lysosome fusion and crinophagy remained unknown (Szenci, 2024).

This study shows that Ykt6 forms a canonical SNAREpin with Syntaxin 13 and Snap29, which is—similarly to the already known Syntaxin 13, Snap29, Vamp7 SNAREpin—critical for crinophagic degradation. Ykt6 localizes to small Lamp1+ (carrier) vesicles and mediates their fusion with SGs, while Vamp7 regulates the fusion of SGs and Arl8+ lysosomes. In summary, this study provides evidence that SG maturation preceding exocytosis and crinophagy requires a series of fusions between SGs and two separate lysosome-related vesicle subpopulations, which are governed by different SNAREpins/SNARE complexe (Szenci, 2024).

This work revealed that Ykt6 acts together with Syntaxin 13 and Snap29 to form a functional SNAREpin. This SNARE complex is required for efficient SG fusion with Lamp1 carrier vesicles, thereby promoting the maturation and crinophagic elimination of SGs. The first vesicle fusions occur just before the bulk secretion of SGs. These early fusions may drive the acidification and inner reorganization of SGs to promote SG maturation. In line with this, SG-lysosome fusion is claimed to cause enhanced secretion in Trpml1^−/− mutant pancreatic acinar cells. The data indicate that the two R-SNAREs Ykt6 and Vamp7 play different roles in regulating the maturation of SGs, because SGs fail to acidify properly without Vamp7, while the silencing of ykt6 did not prevent this (Szenci, 2024).

Moreover, Vamp7 was found to be required for the localization multiple lysosomal markers to maturing glue granules, while Ykt6 only affects the fusion of SGs with Lamp1+ vesicles and it is dispensable for fusion with Arl8+ lysosomes. Maturing SGs are hypothesized to first undergo Vamp7-mediated fusion with Arl8+ lysosomes, which is required for their maturation. The Ykt6-mediated fusions between SGs and Lamp1+ vesicles likely represent a later step of SG maturation. Although the current findings suggest the existence of at least two separate vesicle subpopulations carrying these lysosomal markers (Arl8+ ones and Lamp1+/Arl8− ones), Arl8+ lysosomes possibly also contain Lamp1 (Szenci, 2024).

The coexistence and sequential contribution of multiple lysosomal subpopulations/Lamp1 carriers in distinct steps of SG maturation could be an advantage for secretory cells. Different vesicle subpopulations can act as carriers that deliver different lysosomal membrane proteins and enzymes that are required for lysosome biogenesis. The volume of SGs is enormous compared to these small vesicles, hence the desired concentration of lysosomal proteins in matured SGs or crinosomes could be fine-tuned by a series of membrane fusions with different lysosomal populations. This model is further supported by findings by others, showing that Vamp7 is required for the transport of lysosomal membrane proteins (LMPs, including Lamp1) or the potential role of Ykt6 in lysosomal enzyme transport. Since this study found that the vesicles to which Ykt6 localizes are positive only for Lamp1, but negative for Arl8 and the lysosomal protease Cathepsin L, Ykt6 appears to be required for the delivery of lysosomal membrane proteins such as Lamp1 itself to glue granules. As the highly glycosylated Lamp1 is essential for protecting the lysosomal membrane from acidic internal pH and enzymatic degradation, the Ykt6-mediated delivery of these Lamp1 carrier vesicles to mature SGs could prepare them for the degradative crinosomal fate (Szenci, 2024).

This study has also demonstrated the importance of endosomal contribution to crinosome formation. PI3P+ endosomes are much smaller than SGs and initially form clusters among SGs before secretion60, eventually fusing with the residual, non-secreted mature SGs. One can assume that these endosomal fusions prepare the obsolete SGs for crinophagic breakdown, possibly through the recruitment of Rab7, which is implicated in crinophagic SG-lysosome fusion. Importantly, this study found that these fusions are equally hampered in the absence of either Ykt6 or Vamp7. Thus, the early Ykt6- and Vamp7-mediated vesicle fusions determine the subsequent fate and fusion potential of maturing SGs. Small PI3P+ endosomes that fuse with residual SGs are likely derived from endocytic activity that follows the programmed secretion of SGs. The convergence of secretory, endosomal, and even autophagic routes in lysosomes was also demonstrated in larval Drosophila fat tissue (Szenci, 2024).

Overall, these results refine the model of glue granule maturation and lysosome fusions: SGs probably first acquire the lysosomal small GTPase Arl8 and begin to acidify via fusion by the canonical Vamp7 containing SNAREpin. This primary fusion event engages maturing SGs for subsequent volume-increasing lysosomal fusions that already involve Lamp1+ lysosomes. Ykt6 reaches SGs by forming a SNAREpin with Syntaxin 13 Qa- and Snap29 Qbc-SNAREs to mediate SG-Lamp1 carrier vesicle fusion, and these separate fusion events together promote the maturation and crinophagic degradation of residual glue granules after secretion (Szenci, 2024).

Snazarus and its human ortholog SNX25 modulate autophagic flux

Macroautophagy, the degradation and recycling of cytosolic components in the lysosome, is an important cellular mechanism. It is a membrane-mediated process that is linked to vesicular trafficking events. The sorting nexin (SNX) protein family controls the sorting of a large array of cargoes, and various SNXs impact autophagy. To improve understanding of their functions in vivo, all Drosophila SNXs were screened using inducible RNA interference in the fat body. Significantly, depletion of Snazarus (Snz) led to decreased autophagic flux. Interestingly, altered distribution of Vamp7-positive vesicles was observed with Snz depletion, and the roles of Snz were conserved in human cells. SNX25, the closest human ortholog to Snz, regulates both VAMP8 endocytosis and lipid metabolism. Through knockout-rescue experiments, it was demonstrated that these activities are dependent on specific SNX25 domains and that the autophagic defects seen upon SNX25 loss can be rescued by ethanolamine addition. The presence of differentially spliced forms of SNX14 and SNX25 was detected in cancer cells. This work identifies a conserved role for Snz/SNX25 as a regulator of autophagic flux and reveals differential isoform expression between paralogs (Lauzier, 2022).

Macroautophagy, hereafter termed autophagy, is a crucial homeostatic and stress-responsive catabolic mechanism. Autophagy is characterized by the formation of double-membrane structures, called phagophores, which expand and incorporate cytoplasmic proteins or organelles. These structures ultimately close to form autophagosomes. When mature, the autophagosomes fuse with lysosomes, and autophagosomal content is degraded by lysosomal enzymes and recycled. Hence, autophagy requires an intricate balance between various cellular processes to ensure appropriate cargo selection, and autophagosome formation, maturation and fusion (Lauzier, 2022).

Although the core signaling pathways controlling autophagy induction in response to stress were rapidly described and are now well understood, the molecular mechanisms controlling autophagosome sealing, maturation and fusion were only defined more recently. Findings in yeast and metazoans have shed light on the molecular machinery required for autophagosome-lysosome fusion and its regulation. Although different proteins are involved in autophagosome-vacuole fusion in yeast and autophagosome-lysosome fusion in metazoans, the overarching principle is conserved and requires the presence of specific soluble N-ethylmaleimide-sensitive factor attachment receptors (SNAREs). In metazoans, syntaxin (STX) is recruited to mature autophagosomes by two hairpin regions, where it forms a Qabc complex with synaptosome associated protein 29 (SNAP29). The STX17-SNAP29 complex then forms a fusion-competent complex with lysosome-localized vesicle associated membrane protein (VAMP). More recently, the Qa SNARE YKT6 v-SNARE homolog (YKT6) was also found to mediate autophagosome-lysosome fusion. YKT6 is recruited to mature autophagosomes and associates with SNAP29. The YKT6-SNAP29 complex interacts with the lysosomal R-SNARE STX7 to mediate fusion. These fusion complexes are conserved, and flies also use these proteins for autophagosome-lysosome fusion. However, unlike in human cells, where STX17 and YKT6 act redundantly in parallel pathways, Ykt6 is epistatic to Syx17 and Vamp7 in flies. SNARE functions are supported by other intracellular factors, which ensure their specificity and rapid action. The small Rab GTPases Ras-related protein RAB7 and RAB2 are important determinants of fusion, as lysosome-localized RAB7 and autophagosome-localized RAB2 interact with the tethering homotypic fusion and vacuole protein sorting (HOPS) complex to bring autophagosomes and lysosomes in close proximity and enable SNARE-mediated fusion. Interestingly, a direct interaction has been observed between STX17 and the HOPS complex, favoring autophagosome-lysosome tethering. The lipid composition of autophagosomes and lysosomes is also an important determinant of fusion. Specific phosphoinositides [PtdIns(3)P, PtdIns(3,5)P2, PtdIns(4)P, and PtdIns(4,5)P2] impact fusion through different mechanisms. Low cholesterol levels affect autophagosome tethering to late endosomes/lysosomes, while increased saturated fatty acid levels or a high-fat diet in mice decrease fusion events. Recently, the phosphatidylserine:phosphatidylethanolamine ratio was also demonstrated to affect autophagosome-lysosome fusion (Lauzier, 2022).

It is clear that multiple inputs are integrated to regulate the final step of the autophagic process. Accordingly, trafficking events must properly regulate the trafficking of essential SNAREs involved in autophagosome-lysosome fusion, like VAMP8 and STX7, that also mediate various other membrane fusion events. This is also true for the dynamic regulation of the lipid composition of these organelles, given that inappropriate ratios of specific lipids affect autophagic flux. Hence, defining trafficking regulators coordinating the localization of SNAREs, as well as the lipid composition of autophagosomes and lysosomes, is of paramount importance for better understanding of the dynamic link between trafficking and autophagy (Lauzier, 2022).

One class of endosomal sorting regulators is the sorting nexin (SNX) family. These proteins have phox homology (PX) domains that interact with diverse phosphoinositide species. Many SNXs localize to early endosomes, where they are involved in sorting events. Importantly, a few SNXs play roles in autophagy. SNX18 and SNX4-SNX7 heterodimers control autophagy-related ATG9 trafficking to modulate autophagosome expansion, and SNX5 and SNX6 also indirectly regulate autophagy by modulating cation-independent mannose-6-phosphate receptor sorting, affecting lysosomal functions. In yeast, SNX4 regulates autophagosome-lysosome fusion by controlling endosomal phosphatidylserine levels. These reports highlight the multifaceted roles of SNXs in regulating autophagy. However, SNX involvement in SNARE protein trafficking has not been reported (Lauzier, 2022).

Using Drosophila as a simple system to screen genes involved in autophagy, this study has identified the sorting nexin Snazarus (Snz) and its human ortholog SNX25 as regulators of the localization and lipid metabolism of Vamp7 and VAMP8, respectively. Using RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-generated mutants, as well as ethanolamine supplementation, this study showed that loss of Snz decreases autophagic flux. Importantly, it was shown that this effect is independent of the endoplasmic reticulum (ER) localization of SNX25 and that it affects two independent processes - Vamp7/VAMP8 internalization and lipid homeostasis. Altogether, these findings identify Snz and SNX25 as regulators of autophagic flux (Lauzier, 2022).

This study has uncovered a conserved autophagic function for snz and its ortholog SNX25. Using both RNAi-mediated depletion and CRISPR/Cas9-generated KOs, it was shown that Snz and SNX25 are required for full autophagic flux. The impact on autophagy is unlikely to occur via lysosomal dysfunction, but potentially through a combination of inappropriate Vamp7 (in flies) and VAMP8 (in humans) internalization or trafficking and defective lipid metabolism. Interestingly, the SNX25 PX domain was necessary for VAMP8 uptake, while ER anchoring was dispensable. Furthermore, LC3 accumulation observed upon SNX25 loss could be rescued by SNX25 lacking either its PX/Nexin or ER anchoring domains, and by ETA supplementation. Altogether, the findings uncover the multifaceted effects of SNX25 loss on endocytosis and lipid metabolism, which ultimately affect autophagic flux (Lauzier, 2022).

To further refine the endosomal sorting regulators involved in autophagy, a targeted RNAi screen was performed of SNXs in the fly fat body and monitored autolysosome formation. Unexpectedly, most SNXs tested caused defects in autolysosome acidification. It is believed that this is a consequence of the wide range of cargos sorted or endocytosed by SNXs. The misrouting of specific cargos could directly or indirectly affect lysosomal function and therefore autolysosome acidification or formation. The results also reveal the potential for complementation between SNXs paralogs in mammalian cells, which may explain why autophagy defects were not observed for most SNXs in genome-wide screens (Lauzier, 2022).

SNX14 has three paralogs in mammals - SNX13, SNX19 and SNX25. In neural precursor cells derived from patients with SCAR20, SNX14 loss was associated with autophagosome clearance defects. Conversely, weak effects were observed in dermal fibroblasts from patients. As Drosophila have only a single ortholog of these proteins,it was possible to show through multiple approaches that loss of Snz affected autophagosome clearance and led to autophagosome and autophagic cargo [ref(2)P] accumulation. Data in HeLa cells also indicate defective autophagic flux in SNX14- and SNX25-KO cells. The differences between the current results and findings in patient fibroblasts might be due to differential regulation of either paralog expression or mRNA splicing between cell types. It is worth mentioning that, in HeLa cells, increased SNX14 expression was detected upon SNX25 KO (Fig. 2F). Furthermore, given the complementation of SNX25 KO by SNX14 expression, it is conceivable that SNX25 expression could be differentially modulated in various cell types and be able to rescue SNX14-linked autophagic defects (Lauzier, 2022).

The data indicate defects in the trafficking of Vamp7 and VAMP8 after depletion of Snz and SNX25, respectively. Since the YKT6-SNAP29-STX7 complex can also promote autophagosome-lysosome fusion, it is likely that this complex partially complements the loss of Snz and SNX25, which would explain why their loss did not completely abrogate autophagic flux. Along these lines, differential expression of SNARE complexes between cell types could also account for the variations in penetrance observed between SNX14 studies (Lauzier, 2022).

How exactly Snz/SNX25 regulates Vamp7/VAMP8 endocytosis or trafficking remains to be defined. It was not possible to directly test Vamp7 trafficking in flies; however, ectopic accumulation of GFP:Vamp7 puncta was observed near or at the PM, suggesting a potential uptake defect. To test this more directly, VAMP8 uptake was assessed in SNX25 KO cells. Interestingly, these cells showed decreased VAMP8 internalization that was dependent on the SNX25 PX domain, which interacts with diphosphorylated phosphoinositides like PtdIns(4,5)P2, which is highly abundant at the PM. Defects were not observed in clathrin-dependent or -independent endocytosis, nor were variations in clathrin recruitment at the PM. Hence, it is unlikely that SNX25 depletion results in VAMP8 trafficking defects by affecting PtdIns(4,5)P2 or PtdIns(3,4)P2 dynamics at the PM. Recently, Snz was demonstrated to bridge PM-ER contact sites to modulate LD formation. Therefore, SNX25 may fulfill a similar function in mammals, bridging PM-ER contact sites to favor VAMP8 internalization. A precedent for the involvement of ER-PM contact sites in endocytosis exists; however, it was possible to rescue VAMP8 internalization in SNX25 KO cells with a transgene lacking its ER-anchoring domains, implying that ER-PM proximity is not required for efficient VAMP8 uptake. This notion is consistent with the known requirement of PICALM for VAMP8 uptake. Surprisingly, no defects were detected in PICALM localization in SNX25 or SNX14/SNX25 KO cells, although close proximity between it and overexpressed SNX25 was observed. VAMP8 can also be internalized through a clathrin-independent pathway stimulated by Shiga toxin. This pathway is dependent on lipid organization and might be perturbed in SNX25 KO cells. An earlier study identified SNX25 as a regulator of transforming growth factor β receptor (TGFβR) endocytosis. However, this study erroneously characterized the ΔTM isoform of SNX25 and showed that overexpression of this short isoform increased TGFβR internalization, while SNX25 knockdown decreased uptake. Thus, Snz/SNX25 might affect the endocytosis of multiple cargos, in addition to Vamp7 and VAMP8 (Lauzier, 2022).

It is also worth mentioning that the yeast ortholog of snz and SNX25, MDM1, was originally identified as a regulator of endocytic trafficking, thus other aspects of trafficking could be impaired in Snz/SNX25 mutants and be sensitive to protein expression levels. Although the data illustrate decreased internalization of VAMP8 in SNX25 KO cells, the possibility remains that VAMP8, in addition to its uptake defect, could be misrouted on route to autolysosomes. Decreased colocalization was observed between VAMP8 and CD63 in SNX25 KO cells; therefore, defective trafficking cannot be ruled out. Moreover, co-expression of both SNX25 and VAMP8 led to the re-localization of both proteins to large internal vesicles. This effect required the TM region of SNX25, thus it is conceivable that although the short isoform is sufficient for VAMP8 internalization, the longer ER-associated isoform could regulate the endosomal sorting of VAMP8, through potential inter-organellar contact sites or by modulating lipid metabolism (Lauzier, 2022).

Recent studies have demonstrated important roles for SNX14 in lipid metabolism. SNX14 loss results in saturated fatty acid accumulation and increased sensitivity to lipotoxic stress. Moreover, SNX14, Snz and Mdm1, the yeast ortholog, all regulate LD formation. The functional domains required for SNX14 regulation of LD formation differ from the ones required in SNX25 for VAMP8 uptake; the TM and C-terminal nexin domains of SNX14 are essential for LD localization and regulation, while the PX domain of SNX25 is required for VAMP8 uptake, and its TM domains are dispensable. Interestingly, LD biogenesis, fatty acid trafficking and autophagy are known to intersect. In this context, it is tempting to speculate that Snz and its human orthologs SNX14 and SNX25 could bridge lipid stress and autophagy regulation. Further supporting this hypothesis is the finding that SNX25 loss can be rescued by SNX14 or by either SNX25ΔTM and SNX25ΔPX/Nexin. Moreover, ETA addition, which is predicted to result in higher intracellular phosphatidylethanolamine levels, rescued SNX25 deletion. These rescue experiments highlight that SNX25 loss causes independent phenotypes that culminate in decreased autophagic flux. The effects are likely more potent in flies, since they have a single ortholog and the data show that SNX14 can efficiently rescue SNX25 loss. Concerning the role of SNX25 in lipid metabolism, it is tempting to speculate that it is most probably linked to an effect on lipid saturation and LD biogenesis for four main reasons. First, the C-Nexin region of SNX14 was shown to mediate LD localization, and SNX25 loss could be rescued using a SNX25 mutant deleted of this region, arguing that LD recruitment of SNX25 is dispensable. Second, KO/rescue experiments in HeLa cells were performed in normal growth conditions, where LD biogenesis is minimal, and thus unlikely to affect autophagy. Third, recent findings in U2OS cells identified the PXA region of SNX14 as important in regulating lipid saturation and ER stress in response to saturated lipid accumulation. As the PXA was conserved in the two rescue constructs used for autophagy rescue, it is plausible that SNX25 somehow affects lipid homeostasis and thus autophagosome-lysosome fusion. Moreover, recent findings illustrated the importance of the PE ratio in membrane fusion, and SNX14 deletion leads to increased phosphatidylserine levels as in SNX4 yeast mutants (Ma et al., 2018). This intriguing possibility warrants further studies to identify the specific determinants that mediate the action of SNX25 in endocytosis versus lipid homeostasis (Lauzier, 2022).

Another possibility to consider is that SNX25 may encode ba lipid clustering or transport domain that could help concentrate lipids or move them between organelles in a manner that support functional autophagy. In support of this, recent work using Alphafold2 structural predictions suggest that the Nexin-C and PXA domains of the yeast SNX25 ortholog Mdm1 fold together to create a large spherical domain with a hydrophobic channel that could, in principle, ferry lipids between organelles at organelle contacts. Such a domain could enable SNX25 to localize to various intracellular sites, and cluster and/or transport lipids to support functional autophagy. SNX14 is predicted to contain this domain arrangement as well and this might explain why it can rescue SNX25 loss. In this model, loss of SNX25 would alter lipid homeostasis and subcellular distribution, leading to defects in Vamp7/VAMP8 trafficking and functional autophagy. The molecular details for this process, however, remain to be addressed (Lauzier, 2022).

The observation that various isoforms of SNX14 and SNX25 are expressed in cells is intriguing. This raises the possibility of functional pools of SNX14 and SNX25, with the longer ER-anchored isoform regulating LD biogenesis and the shorter isoforms regulating other processes, like trafficking and autophagy. It is worth noting, however, that although this study provides evidence from ddPCR experiments, it was not possible to demonstrate differential splicing at the protein level because of a lack of isoform-specific antibodies. Isoform expression may be controlled by modulating splicing in response to stress, as has been observed for multiple genes. Alternatively, different transcription factors may favor the expression of certain isoforms. RNA-sequencing datasets from Drosophila do not contain different Snz isoforms, suggesting that a single isoform regulates both LD biogenesis and autophagy (Lauzier, 2022).

In summary, this study has identified a new role for snz and its ortholog SNX25 in autophagy regulation through effects on Vamp7/VAMP8 internalization and lipid metabolism. Moreover, differentially expressed isoforms of SNX14 and SNX25 were described in cancer cells. Based on thesd results and those of previous studies, it is propose that Snz and SNX25 finetune the endocytosis/trafficking of Vamp7 and VAMP8 and potentially regulate the lipid composition of endolysosomes to coordinate the autophagy level with the demands of the cell. It will be interesting to define how these functions differ between various genes and isoforms, and how they are affected by different stressors (Lauzier, 2022).

The R-SNARE Ykt6 is required for multiple events during oogenesis in Drosophila

Ykt6 has emerged as a key protein involved in a wide array of trafficking events, and has also been implicated in a number of human pathologies, including the progression of several cancers. It is a complex protein that simultaneously exhibits a high degree of structural and functional homology, and yet adopts differing roles in different cellular contexts. Because Ykt6 has been implicated in a variety of vesicle fusion events, this study characterized the role of Ykt6 in oogenesis by observing the phenotype of Ykt6 germline clones. Immunofluorescence was used to visualize the expression of membrane proteins, organelles, and vesicular trafficking markers in mutant egg chambers. Ykt6 germline clones have morphological and actin defects affecting both the nurse cells and oocyte, consistent with a role in regulating membrane growth during mid-oogenesis. Additionally, these egg chambers exhibit defects in bicoid and oskar RNA localization, and in the trafficking of Gurken during mid-to-late oogenesis. Finally, it was shown that Ykt6 mutations result in defects in late endosomal pathways, including endo- and exocytosis. These findings suggest a role for Ykt6 in endosome maturation and in the movement of membranes to and from the cell surface (Pokrywka, 2021).

Phosphorylation of Ykt6 SNARE Domain Regulates Its Membrane Recruitment and Activity

Sensitive factor attachment protein receptors (SNARE) proteins are important mediators of protein trafficking that regulate the membrane fusion of specific vesicle populations and their target organelles. The SNARE protein Ykt6 lacks a transmembrane domain and attaches to different organelle membranes. Mechanistically, Ykt6 activity is thought to be regulated by a conformational change from a closed cytosolic form to an open membrane-bound form, yet the mechanism that regulates this transition is unknown. This study identified phosphorylation sites in the SNARE domain of Ykt6 that mediate Ykt6 membrane recruitment and are essential for cellular growth. Using proximity-dependent labeling and membrane fractionation, phosphorylation was found to regulates Ykt6 conversion from a closed to an open conformation. This conformational switch recruits Ykt6 to several organelle membranes, where it functionally regulates the trafficking of Wnt proteins and extracellular vesicle secretion in a concentration-dependent manner. It is proposed that phosphorylation of its SNARE domain leads to a conformational switch from a cytosolic, auto-inhibited Ykt6 to an active SNARE at different membranes (Karuna, 2020).

Ykt6-dependent endosomal recycling is required for Wnt secretion in the Drosophila wing epithelium

Morphogens are important signalling molecules for tissue development and their secretion requires tight regulation. In the wing imaginal disc of flies, the morphogen Wnt/Wingless is apically presented by the secreting cell and re-internalized before final long-range secretion. Why Wnt molecules undergo these trafficking steps and the nature of the regulatory control within the endosomal compartment remain unclear. This study has investigated how Wnts are sorted at the level of endosomes by the versatile v-SNARE Ykt6. Using in vivo genetics, proximity-dependent proteomics and in vitro biochemical analyses, most Ykt6 was shown to be present in the cytosol, but can be recruited to de-acidified compartments and recycle Wnts to the plasma membrane via Rab4-positive recycling endosomes. Thus, a molecular mechanism is proposed by which producing cells integrate and leverage endocytosis and recycling via Ykt6 to coordinate extracellular Wnt levels (Linnemannstons, 2020).

Another longin SNARE for autophagosome-lysosome fusion-how does Ykt6 work?

Formation of the autolysosome involves SNARE-mediated autophagosome-lysosome fusion, which is mediated by a combination of the Qa SNARE STX17 (syntaxin 17), the Qbc SNARE SNAP29 and the R-SNAREs VAMP7/8. 2 very recent reports have now implicated another R-SNARE with a longin domain, YKT6, in this fusion process. Interestingly, these reports painted two different pictures of YKT6's involvement. Studies in HeLa cells indicated that YKT6, acting independently of STX17, could form a separate SNARE complex with SNAP29 and another Qa SNARE to mediate autophagosome-lysosome fusion. Conversely, work in Drosophila larvae fat cells showed that while Ykt6 could form a SNARE complex with Snap29 and Syx17/Stx17, it is readily outcompeted by lysosomal Vamp7 in this regard. Moreover, its activity in autophagosome-lysosome fusion is not impaired by mutation of the supposedly critical ionic zero-layer residue from R to Q. In this regard, YKT6 may therefore act in a noncanonical way to regulate fusion. Here, we ponder on the fresh mechanistic perspectives on the final membrane fusion step of macroautophagy/autophagy offered by these new findings. Further, we propose another possible mechanism as to how YKT6 might act, which may provide some reconciliation to the differences observed. Abbreviations: LD: longin domain (Yong, 2019).

The multi-functional SNARE protein Ykt6 in autophagosomal fusion processes

Autophagy is a degradative pathway in which cytosolic material is enwrapped within double membrane vesicles, so-called autophagosomes, and delivered to lytic organelles. SNARE (Soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins are key to drive membrane fusion of the autophagosome and the lytic organelles, called lysosomes in higher eukaryotes or vacuoles in plants and yeast. Therefore, the identification of functional SNARE complexes is central for understanding fusion processes and their regulation. The SNARE proteins Syntaxin 17, SNAP29 and Vamp7/VAMP8 are responsible for the fusion of autophagosomes with lysosomes in higher eukaryotes. Recent studies reported that the R-SNARE Ykt6 is an additional SNARE protein involved in autophagosome-lytic organelle fusion in yeast, Drosophila, and mammals. These current findings point to an evolutionarily conserved role of Ykt6 in autophagosome-related fusion events. Here, we briefly summarize the principal mechanisms of autophagosome-lytic organelle fusion, with a special focus on Ykt6 to highlight some intrinsic features of this unusual SNARE protein (Kriegenburg, 2019).

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

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. This study demonstrates that, unexpectedly, one more SNARE, Ykt6, is also required for autophagosome clearance in Drosophila. 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 this study demonstrates 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 (Takats, 2018).

VAMP3/Syb and YKT6 are required for the fusion of constitutive secretory carriers with the plasma membrane

The cellular machinery required for the fusion of constitutive secretory vesicles with the plasma membrane in metazoans remains poorly defined. To address this problem a powerful, quantitative assay was developed for measuring secretion, and it was used in combination with combinatorial gene depletion studies in Drosophila cells. This has allowed identification of at least three SNARE complexes mediating Golgi to PM transport (STX1, SNAP24/SNAP29 and Syb; STX1, SNAP24/29 and YKT6, STX4, SNAP24 and Syb). RNAi mediated depletion of YKT6 and VAMP3 in mammalian cells also blocks constitutive secretion suggesting that YKT6 has an evolutionarily conserved role in this process. The unexpected role of YKT6 in plasma membrane fusion may in part explain why RNAi and gene disruption studies have failed to produce the expected phenotypes in higher eukaryotes (Gordon, 2017).

Constitutive secretion delivers newly synthesised proteins and lipids to the cell surface and is essential for cell growth and viability. This pathway is required for the exocytosis of molecules such as antibodies, cytokines and extracellular matrix components so has both significant physiological and commercial importance. The majority of constitutive secreted proteins are synthesised at the endoplasmic reticulum, pass through the Golgi, and are transported to the cell surface in small vesicles and tubules which fuse with the plasma membrane. Constitutive secretory vesicles are not stored within the cell and do not require a signal to trigger their fusion with the plasma membrane which is in contrast to dense core secretory granules or synaptic vesicles. In some cell types, such as MDCK cells and macrophages, there is evidence that constitutive secretory cargo passes through a endosomal intermediate on its way to the cell surface. However, in non-polarised cells endosomal intermediates do not appear to play a major role in this pathway (Gordon, 2017).

Vesicle fusion is driven by a family of molecules known as SNAREs. SNARE are generally small (14-42kDa), C-terminally anchored proteins that have a highly conserved region termed the SNARE motif that has the ability to interact with other SNAREs. For membrane fusion to occur, SNAREs on opposing membranes must come together and their SNARE motifs zipper up to form a SNARE complex. Detailed characterisation of the neuronal SNARE complex (syntaxin 1A/VAMP2/SNAP25) required for synaptic vesicle fusion has provided a mechanistic framework for understanding the function of SNAREs. There are 38 SNAREs encoded in the human genome and they can be classified as either R or Q-SNAREs depending on the presence of a conserved arginine or glutamine in their SNARE motif. Q-SNAREs can be further subdivided into Qa, Qb and Qc SNAREs based on their homology to syntaxin and SNAP25. A typical fusogenic SNARE complex will contain four SNARE motifs (Qa, Qb, Qc and R). Qbc-SNAREs such as SNAP23, 25, 29 and 47 contribute two SNARE motifs to the SNARE complex. R-SNAREs can also be further classified as either longin or brevin type SNAREs. Longin type R-SNAREs contain a longin type fold and are found in all eukaryotes and while brevin type SNAREs are less widely conserved across species (Gordon, 2017).

Over the past twenty years significant progress has been made defining the SNARE complexes required for the majority of intracellular transport steps within eukaryotic cells. In addition, there are an increasing number of examples where the SNARE complexes required for the secretion of specific cargo such as Wnt, TNF and IL-6 have been identified. However, these proteins are not delivered directly to the cell surface from the TGN but pass through an endosomal compartment. Many labs have attempted to identify the machinery which drive the fusion of constitutive secretory vesicles with the plasma membrane and on the whole very little progress has been made. This in part may be due to the fact that there are multiple routes to the cell surface from the Golgi and redundancy in the fusion machinery. If just the R-SNAREs are considered, the human genome encodes seven post-Golgi SNAREs and a typical mammalian cell line can express at least five R-SNAREs so disruption of just one R-SNARE is unlikely to block secretion if they are functionally redundant. To overcome this problem SNARE function was characterized in Drosophila cells , as they have a simpler genome with less redundancy. The Drosophila genome encodes 26 SNAREs with 16 of them predicted to be localised to post-Golgi membranes based on their homology to mammalian SNAREs. The complexity is reduced even further as Drosophila cell lines just express two post-Golgi R-SNAREs, Syb and VAMP7 (based on publically available microarray data generated by the modENCODE project) (Gordon, 2017).

This study has developed a novel, quantitative assay for measuring constitutive secretion based on a reporter cell line that can be effectively used to monitor secretion by flow cytometry, immunoblotting and fluorescence microscopy. Depletion of known components of the secretory pathway in Drosophila cells (STX5, SLH and ROP) causes robust blocks in ER to Golgi and Golgi to plasma membrane transport, therefore validating this approach. As predicted, there is redundancy in the post-Golgi SNAREs and multiple SNAREs must be depleted to obtain robust blocks in secretion. This study has detected strong negative genetic interactions between Drosophila STX1 and STX4, SNAP24 and SNAP29, STX1 and Syb, and SNAP24 and Syb. A novel and unexpected genetic interaction was detected between Syb and YKT6. Depletion of YKT6 and VAMP3 in mammalian cells also causes a robust block in secretion indicating that this negative genetic interaction is conserved across species and provides evidence that these two R-SNAREs function in the late secretory pathway (Gordon, 2017).

Using well characterised targets (STX5, SLY1 and ROP) this study has validated the system and the assay was shown to be capable of differentiating blocks in ER to Golgi and Golgi to plasma membrane transport based on proteolytic processing and accumulation of the secretory cargo in post-Golgi transport vesicles. The experimental data suggests that there are at least three fusion complexes operating at the Drosophila PM. The first complex consists of STX1, SNAP24/29 and Syb. The second complex consists of STX4, SNAP24/29 and Syb. The third complex consists of STX1, SNAP24 and YKT6. The reason the possibility of a STX4, SNAP24/29, YKT6 complex was excluded is because depletion of both STX1 and Syb led to a complete block in secretion. Indicating that STX4 and YKT6 are unable to form a SNARE complex that can substitute for the loss of STX1 and Syb. Genetic interaction data also suggests that SNAP29 is unable to substitute for the loss of SNAP24 under conditions when both SNAP24 and Syb are depleted. This data suggests that the third SNARE complex specifically consists of STX1, SNAP24 and YKT6. At present it is unclear whether these SNARE complexes define parallel pathways to the plasma membrane or simply reflect the ability of these SNAREs to substitute with each other (Gordon, 2017).

The most striking observation in this study is that an unexpected role for YKT6 in the fusion of secretory carriers with the plasma membrane was uncovered. Depletion of YKT6 and Syb/VAMP3 in combination causes a complete block in secretion and leads to an accumulation of post-Golgi transport vesicles within Drosophila cells. YKT6 is a lipid anchored R-SNARE that has been shown to function on many pathways including ER to Golgi transport, intra-Golgi transport, endosome-vacuole fusion, endosome to Golgi transport and exosome fusion with the plasma membrane. YKT6 actively cycles on and off membranes in a palmitoylation dependant manner so potentially it is well suited to function on a wide variety of intracellular pathways. Due to the promiscuous nature of YKT6 some caution must be taken when interpreting rhe functional data. It is possible that loss of YKT6 may be indirectly affecting post-Golgi transport and fusion at the plasma membrane. However, the simplest interpretation of this data is YKT6 is directly involved in this process as this study was able to biochemically detect an interaction between YKT6 and STX1 (Gordon, 2017).

Using the knowledge obtained from the Drosophila system, the role of R-SNAREs in constitutive secretion in mammalian cells was reexamined. As previously reported, depletion of VAMP3 and other post-Golgi R-SNAREs did not perturb secretion in HeLa cells. However, depletion of VAMP3 and YKT6 in combination caused a complete block in secretion. This data suggests that YKT6 and VAMP3 may be functioning in the fusion of secretory carriers with the plasma membrane in mammalian cells. Significant efforts were made to localise endogenous YKT6 and VAMP3 on post-Golgi secretory carriers. However, the attempts have been hampered by the fact the endogenus YKT6 is expressed at very low levels and over expressed YKT6 does not target correctly to membranes and remains cytoplasmic (Gordon, 2017).

As expected, there is redundancy in the Q-SNAREs required for the fusion of secretory carriers with the plasma membrane. However, it is clear that certain SNAREs have a more prominent role in this process. The main Q-SNAREs at the Drosophila plasma membrane are STX1 and STX4. Depletion of STX1 causes a partial block in secretion while depletion of STX4 does not. It is unclear why STX1 is the favoured Qa-SNARE. It could simply be that STX1 is more abundant than STX4 or has a higher affinity for the R-SNARE on the vesicle. It may also reflect the route by which the synthetic cargo traffics to the cell surface. This study also observed redundancy between the Qbc-SNAREs SNAP24 and SNAP29 (orthologues of Sec9). A complete block in secretion is detected when both are depleted. It has previously been shown that SNAP29 interacts with STX1. However, the complexes it forms are not SDS-resistant suggesting that they may not be fusogenic (Gordon, 2017).

A potential problem with gene disruption and RNAi mediated depletion studies is compensation by other genes in the same family. For example, VAMP2 and 3 are upregulated in certain tissues of the VAMP8 knockout mouse and VAMP3 is upregulated in VAMP2 deficient chromafin cells isolated from VAMP2 null mice. Based on immunoblotting data this study did not observe any compensation between R-SNAREs when they are depleted using RNAi in Drosophila cells. No evidence was seen of this in previous work performed in HeLa cells. It was initially thought that STX1 and STX4 were being upregulated in STX5 and Syb depleted cells based on immunoblotting. However, when the samples were directly prepared in Laemmli sample buffer, rather than a TX100 based extraction buffer, no difference in the levels of these SNAREs was observed. It is possible that the change in extractability may be caused by an alteration in the localisation of the Q-SNAREs from TX100 insoluble micro-domains at the plasma membrane. However, this hypothesis was not tested. To directly assess changes in gene expression during the RNAi experiments the mRNA levels were measured of several SNAREs using RT-PCR. Depletion of STX1 leads to an upregulation of STX4 and Syb. However, no significant change was observed in the protein level of these SNAREs by immunoblotting. Thus it is unclear how significant these changes are. In the future, it will be interesting to determine how the expression levels of SNAREs, which function on the same pathway, are co-ordinated and regulated (Gordon, 2017).

To validate the genetic interaction data a published S. cerevisiae proliferation-based genetic interaction map was have interrogated to determine if the yeast homologues share similar genetic interactions to those observed in Drosophila cells (under the assumption that constitutive secretion is essential for growth). Negative genetic interactions were observed between Drosophila STX1 and STX4, STX1 and Syb, Syb and SNAP24, SNAP24 and SNAP29, YKT6 and Sec22b and Syb and YKT6. Similar genetic interactions were also observed in S. cerevisiae indicating that the data generated from Drosophila cells is physiologically relevant and the genetic interactions are evolutionary conserved. Importantly the homologues of YKT6 and Syb/VAMP3 were also found to genetically interact in yeast (YKT6 and SNC2) (Gordon, 2017).

In summary, this study has identified the SNARE complexes required for the fusion of constitutive secretory vesicles with the plasma membrane in Drosophila cells. This study has uncovered a novel role for YKT6 in the fusion of secretory vesicles with the plasma membrane which is conserved from yeast to man. This observation may in part explain why RNAi and gene disruption studies in higher eukaryotes have failed to yield the expected phenotypes. In the future, it should be possible to use the secretion assay in combination with SNARE depletion as a tool to further dissect the post-Golgi pathways involved in secretion and generate post-Golgi secretory carriers for proteomic profiling (Gordon, 2017).

Active Wnt proteins are secreted on exosome

Wnt signalling has important roles during development and in many diseases. As morphogens, hydrophobic Wnt proteins exert their function over a distance to induce patterning and cell differentiation decisions. Recent studies have identified several factors that are required for the secretion of Wnt proteins; however, how Wnts travel in the extracellular space remains a largely unresolved question. This study shows that Wnts are secreted on exosomes both during Drosophila development and in human cells. Exosomes carry Wnts on their surface to induce Wnt signalling activity in target cells. Together with the cargo receptor Evi/WIs, Wnts are transported through endosomal compartments onto exosomes, a process that requires the R-SNARE Ykt6. This study demonstrates an evolutionarily conserved functional role of extracellular vesicular transport of Wnt proteins (Gross, 2012).

The present study demonstrated by biochemical and genetic approaches that a portion of functional Wnts is secreted on exosomes. The discrepancy between the hydrophobic properties of Wnts and their trafficking over longer distances has been a puzzling observation, leading to different hypotheses on how Wnts disperse. The results demonstrate that Wnts are secreted on exosomes in vivo and ex vivo. These secreted microvesicles originate through inward budding in multivesicular bodies (MVBs) and influence intercellular trafficking. Several lines of evidence support these conclusions. First, by biochemical fractionation it was found Wnt co-segregating with exosomes derived from mammalian and Drosophila cells. Ultrastructural analysis demonstrated that Wg is found on the outer membrane of exosomes. Second, it was demonstrate that Wnts are shuttled to MVBs by their cargo receptor Evi; this step is impaired by inhibition of MVB maturation or depletion of the ESCRT-0 complex, which mediates cargo recognition of exosomal proteins. Third, it was shown that a fraction of extracellular Wg co-localizes with several exosomal markers in vivo. Fourth, Ykt6 was identified as a protein required for secretion of Wnts and exosomal proteins. The results argue for an alternative route of Wnt secretion in vivo that is independent of lipoprotein particles. The biochemical fractionation method differs from the parameters used to biochemically isolate lipoprotein particles; in addition, lipoprotein particles were not found by ultrastructural analysis in the preparations. Taken together, it is believed that this study provides solid evidence for a Wnt secretion route through exosomes (Gross, 2012).

Ykt6 belongs to the longin type of R-SNARE involved in various trafficking events. Studies in yeast have suggested that Ykt6 localizes to the Golgi and to endosomal and vacuolar membranes. This study shows that in Drosophila Ykt6 does not affect secretion of transmembrane proteins such as Ptc and Fmi, and that on loss of Ykt6 in HeLa cells Evi accumulates in early endosomes but not in the Golgi. This finding is consistent with the role of Ykt6 in early/recycling endosomes described in mammalian cells; however, further functional roles for Ykt6 might exist and might also be modulated in a cell-type-specific manner (Gross, 2012).

Interestingly, the results imply that the retromer complex acts upstream of MVB sorting of Evi-Wnt complexes, as demonstrated by the effect of ESCRT and Ykt6 RNAi on secretion of Wnt/Evi and the exosomal marker CD81. The retromer-SNX3 sorting route might decide whether unloaded Evi is recycled to the Golgi or whether Evi-Wnt complexes are packaged onto exosomes. This is supported by the finding that depletion of retromer leads to lysosomal sorting, thereby reducing the pool of Evi as well as inhibiting the secretion of Wnts on exosomes. It has been shown that SNX3 together with HGS functions in sorting and membrane invagination at the MVB level. In yeast, SNX3, similar to Ykt6, retrieves cargo receptors to Golgi before homotypic fusion of vacuoli. Thus, it is tempting to speculate that a retromer-SNX3-dependent step retrieves empty Evi to Golgi, whereas Wnts (and Evi) are packaged onto exosomes for functional release (Gross, 2012).

Different extracellular forms of Wnts might not be mutually exclusive. The results show that removal of vesicle-bound Wnt only partially reduced Wnt activity, implying that other secretion routes exist, possibly through the direct release from the plasma membrane, which might have different extracellular trafficking properties (Gross, 2012).

It is tempting to speculate that morphogens provide spatial information in other forms than concentrations of signalling molecules, involving a 'digital' rather than 'analogue' mode of encoding signals. Different packaging modes of Wnt molecules and their control in Wnt-producing cells might have a profound effect on spreading and signalling properties of morphogens (Gross, 2012).

Homozygous missense variants in YKT6 result in loss of function and are associated with developmental delay, with or without severe infantile liver disease and risk for hepatocellular carcinoma

YKT6 plays important roles in multiple intracellular vesicle trafficking events but has not been associated with Mendelian diseases. This study reports 3 unrelated individuals with rare homozygous missense variants in YKT6 who exhibited neurological disease with or without a progressive infantile liver disease. The variants were We modeled in Drosophila. Wild-type and variant genomic rescue constructs were generated of the fly ortholog dYkt6, and their ability in rescuing the loss-of-function phenotypes were compared in mutant flies. A dYkt6^KozakGAL4 allele was generated to assess the expression pattern of dYkt6. Two individuals are homozygous for YKT6 [NM_006555.3:c.554A>:G p.(Tyr185Cys)] and exhibited normal prenatal course followed by failure to thrive, developmental delay, and progressive liver disease. Haplotype analysis identified a shared homozygous region flanking the variant, suggesting a common ancestry. The third individual is homozygous for YKT6 [NM_006555.3:c.191A>:G p.(Tyr64Cys)] and exhibited neurodevelopmental disorders and optic atrophy. Fly dYkt6 is essential and is expressed in the fat body (analogous to liver) and central nervous system. Wild-type genomic rescue constructs can rescue the lethality and autophagic flux defects, whereas the variants are less efficient in rescuing the phenotypes. The YKT6 variants are partial loss-of-function alleles, and the p.(Tyr185Cys) is more severe than p.(Tyr64Cys) (Ya, 2024). <

Human YKT6 forms priming complex with STX17 and SNAP29 to facilitate autophagosome-lysosome fusion

Autophagy is crucial for degrading and recycling cellular components. Fusion between autophagosomes and lysosomes is pivotal, directing autophagic cargo to degradation. This process is driven by STX17-SNAP29-VAMP8 and STX7-SNAP29-YKT6 in mammalian cells. However, the interaction between STX17 and YKT6 and its significance remain to be revealed. This study, challenges the notion that STX17 and YKT6 function independently in autophagosome-lysosome fusion. YKT6, through its SNARE domain, forms a complex with STX17 and SNAP29 on autophagosomes, enhancing autophagy flux. VAMP8 displaces YKT6 from this complex, leading to the formation of the fusogenic complex STX17-SNAP29-VAMP8. tablehe YKT6-SNAP29-STX17 complex facilitates both lipid and content mixing driven by STX17-SNAP29-VAMP8, suggesting a priming role of YKT6 for efficient membrane fusion. These results provide a potential regulation mechanism of autophagosome-lysosome fusion, highlighting the importance of YKT6 and its interactions with STX17 and SNAP29 in promoting autophagy flux (Zheng, 2024). <

ULK1-mediated phosphorylation regulates the conserved role of YKT6 in autophagy

Autophagy is a catabolic process during which cytosolic material is enwrapped in a newly formed double-membrane structure called the autophagosome, and subsequently targeted for degradation in the lytic compartment of the cell. The fusion of autophagosomes with the lytic compartment is a tightly regulated step and involves membrane-bound SNARE proteins. These play a crucial role as they promote lipid mixing and fusion of the opposing membranes. Among the SNARE proteins implicated in autophagy, the essential SNARE protein YKT6 is the only SNARE protein that is evolutionarily conserved from yeast to humans. This study shows that alterations in YKT6 function, in both mammalian cells and nematodes, produce early and late autophagy defects that result in reduced survival. Moreover, mammalian autophagosomal YKT6 is phospho-regulated by the ULK1 kinase, preventing premature bundling with the lysosomal SNARE proteins and thereby inhibiting autophagosome-lysosome fusion. Together, these findings reveal that timely regulation of the YKT6 phosphorylation status is crucial throughout autophagy progression and cell survival (Sanchez-Martin, 2023).

Ykt6 forms a SNARE complex with syntaxin 5, GS28, and Bet1 and participates in a late stage in endoplasmic reticulum-Golgi transport

The yeast SNARE Ykt6p has been implicated in several trafficking steps, including vesicular transport from the endoplasmic reticulum (ER) to the Golgi, intra-Golgi transport, and homotypic vacuole fusion. The functional role of its mammalian homolog (Ykt6) has not been established. Using antibodies specific for mammalian Ykt6, it was revealed that it is found mainly in Golgi-enriched membranes. Three SNAREs, syntaxin 5, GS28, and Bet1, are specifically associated with Ykt6 as revealed by co-immunoprecipitation, suggesting that these four SNAREs form a SNARE complex. Double labeling of Ykt6 and the Golgi marker mannosidase II or the ER-Golgi recycling marker KDEL receptor suggests that Ykt6 is primarily associated with the Golgi apparatus. Unlike the KDEL receptor, Ykt6 does not cycle back to the peripheral ER exit sites. Antibodies against Ykt6 inhibit in vitro ER-Golgi transport of vesicular stomatitis virus envelope glycoprotein (VSVG) only when they are added before the EGTA-sensitive stage. ER-Golgi transport of VSVG in vitro is also inhibited by recombinant Ykt6. In the presence of antibodies against Ykt6, VSVG accumulates in peri-Golgi vesicular structures and is prevented from entering the mannosidase II compartment, suggesting that Ykt6 functions at a late stage in ER-Golgi transport. Golgi apparatus marked by mannosidase II is fragmented into vesicular structures in cells microinjected with Ykt6 antibodies. It is concluded that Ykt6 functions in a late step of ER-Golgi transport, and this role may be important for the integrity of the Golgi complex (Zhang, 2001).

References

Search PubMed for articles about Drosophila Ykt6

Search PubMed for articles about Drosophila Ykt6

Gordon, D. E., Chia, J., Jayawardena, K., Antrobus, R., Bard, F., Peden, A. A. (2017). VAMP3/Syb and YKT6 are required for the fusion of constitutive secretory carriers with the plasma membrane. PLoS genetics, 13(4):e1006698 PubMed ID: 28403141

Gross, J. C., Chaudhary, V., Bartscherer, K. and Boutros, M. (2012). Active Wnt proteins are secreted on exosomes. Nat Cell Biol 14: 1036-1045. PubMed ID: 22983114

Karuna, M. P., Witte, L., Linnemannstoens, K., Choezom, D., Danieli-Mackay, A., Honemann-Capito, M., Gross, J. C. (2020). Phosphorylation of Ykt6 SNARE Domain Regulates Its Membrane Recruitment and Activity. Biomolecules, 10(11) PubMed ID: 33207719

Kriegenburg, F., Bas, L., Gao, J., Ungermann, C., Kraft, C. (2019). The multi-functional SNARE protein Ykt6 in autophagosomal fusion processes. Cell cycle (Georgetown, Tex), 18(6-7):639-651 PubMed ID: 30836834

Lauzier, A., Bossanyi, M. F., Larcher, R., Nassari, S., Ugrankar, R., Henne, W. M., Jean, S. (2022). Snazarus and its human ortholog SNX25 modulate autophagic flux. J Cell Sci, 135(5) PubMed ID: 34821359

Linnemannstons, K., Witte, L., Karuna, M. P., Kittel, J. C., Danieli, A., Muller, D., Nitsch, L., Honemann-Capito, M., Grawe, F., Wodarz, A., Gross, J. C. (2020). Ykt6-dependent endosomal recycling is required for Wnt secretion in the Drosophila wing epithelium. Development (Cambridge, England), 147(15) PubMed ID: 32611603

Ma, M., Ganapathi, M., Zheng, Y., Tan, K. L., Kanca, O., Bove, K. E., Quintanilla, N., Sag, S. O., Temel, S. G., LeDuc, C. A., McPartland, A. J., Pereira, E. M., Shen, Y., Hagen, J., Thomas, C. P., Nguyen Galván, N. T., Pan, X., Lu, S., Rosenfeld, J. A., Calame, D. G., Wangler, M. F., Lupski, J. R., Pehlivan, D., Hertel, P. M., Chung, W. K., Bellen, H. J. (2024). Homozygous missense variants in YKT6 result in loss of function and are associated with developmental delay, with or without severe infantile liver disease and risk for hepatocellular carcinoma. Genet Med, 26(7):101125 PubMed ID: 38522068

Pokrywka, N. J., Bush, S. and Nick, S. E. (2021). The R-SNARE Ykt6 is required for multiple events during oogenesis in Drosophila. Cells Dev 169: 203759. PubMed ID: 34856414

Sanchez-Martin, P., Kriegenburg, F., Alves, L., Adam, J., Elsaesser, J., Babic, R., Mancilla, H., Licheva, M., Tascher, G., Munch, C., Eimer, S., Kraft, C. (2023). ULK1-mediated phosphorylation regulates the conserved role of YKT6 in autophagy. J Cell Sci, 136(3) PubMed ID: 36644903

Szenci, G, GlatzG., Takaes, S., Juhasz, G. (2024). The Ykt6-Snap29-Syx13 SNARE complex promotes crinophagy via secretory granule fusion with Lamp1 carrier vesicles. Sci Rep, 14(1):3200 PubMed ID: 38331993

Takats, S., Glatz, G., Szenci, G., Boda, A., Horvath, G. V., Hegedus, K., Kováas, A. L., Juhasz, G. (2018). Non-canonical role of the SNARE protein Ykt6 in autophagosome-lysosome fusion. PLoS genetics, 14(4):e1007359 PubMed ID: 29694367

Yong, C. Q. Y., Tang, B. L. (2019). Another longin SNARE for autophagosome-lysosome fusion-how does Ykt6 work? Autophagy, 15(2):352-357 PubMed ID: 30290706

Zhang, T. and Hong, W. (2001). Ykt6 forms a SNARE complex with syntaxin 5, GS28, and Bet1 and participates in a late stage in endoplasmic reticulum-Golgi transport. J. Biol. Chem. 276(29): 27480-7. 11323436

Zheng, D., Tong, M., Zhang, S., Pan, Y., Zhao, Y., Zhong, Q., Liu, X. (2024). Human YKT6 forms priming complex with STX17 and SNAP29 to facilitate autophagosome-lysosome fusion. Cell Rep, 43(2):113760 PubMed ID: 38340317

date revised: 20 April, 2025

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