InteractiveFly: GeneBrief

Synaptosomal-associated protein 29kDa: Biological Overview | References


Gene name - Synaptosomal-associated protein 29kDa

Synonyms -

Cytological map position - 60A3-60A3

Function - signaling

Keywords - interacts with SNARE proteins - required for protein trafficking and for proper Golgi apparatus morphology - promotes kinetochore assembly during mitosis - Snap29 mutant imaginal discs exhibit impairment of a late step of autophagy

Symbol - Snap29

FlyBase ID: FBgn0034913

Genetic map position - chr2R:23,843,483-23,844,763

NCBI classification - SNARE_SNAP29N: N-terminal SNARE motif of SNAP29, C-terminal SNARE motif of SNAP29, a member of the Qb/Qc subfamily of SNARE proteins

Cellular location - cytoplasmic



NCBI links: EntrezGene, Nucleotide, Protein
BIOLOGICAL OVERVIEW

The kinetochore is an essential structure that mediates accurate chromosome segregation in mitosis and meiosis. While many of the kinetochore components have been identified, the mechanisms of kinetochore assembly remain elusive. This study identified a novel role for Snap29, an unconventional SNARE, in promoting kinetochore assembly during mitosis in Drosophila and human cells. Snap29 localizes to the outer kinetochore and prevents chromosome mis-segregation and the formation of cells with fragmented nuclei. Snap29 promotes accurate chromosome segregation by mediating the recruitment of Knl1 at the kinetochore and ensuring stable microtubule attachments. Correct Knl1 localization to kinetochore requires human or Drosophila Snap29, and is prevented by a Snap29 point mutant that blocks Snap29 release from SNARE fusion complexes. Such mutant causes ectopic Knl1 recruitment to trafficking compartments. It is proposed that part of the outer kinetochore is functionally similar to membrane fusion interfaces (Morelli, 2016).

Cell division relies on organization of a microtubule (MT) spindle to which replicated chromosomes become attached for equal segregation. Defective MT attachment to kinetochores (KTs) leads to chromosome mis-segregation and formation of fragmented nuclei after cell division. These events have been proposed to contribute to the genome instability observed in many cancers, indicating that control of MT attachment is a major tumor suppressing process (Morelli, 2016).

The molecular nature of the outer KT, the structure that mediates MT attachment, has been studied extensively. MTs are engaged and stabilized by the Knl1, Mis12, and Ndc80 complexes, together referred to as the KMN network. The KMN network also holds in place the Rod-Zw10-Zwilch (RZZ) complex and spindle assembly checkpoint (SAC) proteins, which are important for signaling incomplete attachment. In mammalian cells, MT attachment is further assisted by the KNL1-interactor ZWINT and by the SKA complex, which associates with curved MT ends at KTs (Morelli, 2016).

In sheer contrast with the extensive molecular knowledge of the outer KT, much less is known about the steps that regulate its assembly. In Drosophila, a widely used metazoan model system for KT studies, part of the Mis12 complex resides at the KT throughout the cell cycle, while the rest of the outer KT is created de novo in early prophase, by stepwise addition of components. The earliest components added to the outer KT in early prophase appears to be Knl1, followed by the Ndc80 complex (Venkei, 2012), both of which are recruited from unknown cellular locales, and SAC components, such as Mad1 and Mad2, which are recruited from nuclear pores (Morelli, 2016).

Unexpectedly, it has been recently found that the Drosophila SNARE protein Snap29 can be isolated from cell extracts together with multiple components of the KMN network. These are the Drosophila Knl1 ortholog Spc105R, three out of four components of the Ndc80 complex (Nuf2 and the Drosophila Spc24 ortholog Kmn2) as well as three of the four subunits of the Mis12 complex (Mis12, Nnf1b, and the Drosophila Nsl1 ortholog Kmn1) (Morelli, 2014). SNARE (Soluble NSF Attachment REceptor) proteins (SNAREs) are part of the conserved coiled-coil machinery that brings membranes in close proximity during trafficking, a prerequisite for most membrane fusion events. The Synaptosomal-Associated Protein (SNAP) family of SNAREs in metazoans includes Snap25, Snap23, and Snap29, which are composed by two SNARE domains, separated by a linker region. The first two proteins are membrane-associated and control synaptic transmission and a wide range of non-neuronal membrane fusion processes, respectively. In contrast, Snap29 only transiently associates with membranes and contains an acidic NPF motif that mediates its association with endocytic factors (Steegmaier, 1998; Rotem-Yehudar, 2001; Su, 2001). Such unconventional features, which are exclusive of Snap29 among the SNARE proteins, predict involvement in a versatile set of membrane trafficking processes, in line with reports in the literature. Consistent with this, Snap29 also controls fusion of autophagosomes with endo-lysosomes in Drosophila and human cells, together with the SNAREs syntaxin17 (Syx17) and Vamp7 (VAMP8 in human cells) (Morelli, 2016).

Despite involvement of Snap29 in multiple trafficking pathways in interphase, a possible function during cell division has not been explored. This study investigated whether Snap29 localizes and acts at the KT in cells and tissues. The data identify a novel step of KT formation that is conserved and supports tissue formation (Morelli, 2016).

The data uncover an essential and conserved step of KT formation that occurs in prophase and requires Snap29. Such step controls localization of Knl1 (and ZWINT in human cells) to KTs. Snap29 and the RZZ components Rod and Zw10 are known to act in membrane transport between the Golgi apparatus and the ER, and the RZZ complex shares similarity with ER tethering complexes. Strikingly, the autophagosome, which depends on Snap29 for fusion to lysosomes, is formed de novo using Golgi and ER components and engages MTs for dynein-directed transport to lysosomes, evoking tantalizing similarities between aspects of membrane trafficking and KT formation. Overall, the current data support the possibility that Snap29, Knl1, and the RZZ complex might act at the KT similarly to tethering and fusion complexes existing on membranes, which need to stabilize MTs during trafficking events. Such scenario might imply common ancestry, and underscored also the existence of protozoa that divide with KTs associated with the nuclear membrane that is itself attached to MT fibers (Morelli, 2016).

Ectopic recruitment of Knl1 and possibly RZZ and SKA complex components at sites of SNAP29 Q1 Q2 trapping predicts that the trafficking and KT functions of Snap29 are interconnected, perhaps because of the existence of a common cellular pool of Snap29. A possibility that awaits further investigation is that SNARE domains of Snap29 interact with KT proteins directly. Interestingly, the C-terminal part of Knl1, Zwint, and Mis12 and Nnf1 all contain multiple coiled-coil regions. These are placed at the interaction surface between the Mis12, Ndc80, and Knl1 complexes, exactly where Snap29 is seen located by super-resolution microscopy. In mammalian cells, the Snap29 paralog Snap25 binds Zwint, and Snap29 has been found in Zwint immunoprecipitations. Thus, Snap29 could dock to the Mis12 complex with a SNARE domain and could stabilize interactions with Knl1 and Zwint with the second C-terminal SNARE domain. Interestingly, no Zwint homolog has been found in Drosophila, suggesting that in flies Snap29 could substitute for Zwint. The ability of KNL1 to interact with a SNAP29 that cannot be released from SNARE complex, both suggest that the interaction of KNL1 with SNAP29 might occur on the side of the SNARE domain that is not occupied by a syntaxin or a Vamp. These data also suggest that SNAP29 could act on Knl1 also prior to nuclear entry and KT localization (Morelli, 2016).

The evidence in vivo indicates that SNAP29 function could support tissue development by ensuring faithful chromosome segregation and that such activity is crucial in cells that become resistant to apoptosis. Based on this, it is predicted that loss of SNAP29 could be selected in cancers with highly unstable genomes. In addition, rare congenital syndromes such as CEDNIK, Roberts syndrome, and primary microcephaly (MCPH) are caused by mutations in Snap29 and other genes encoding proteins that regulate mitosis, respectively, overall suggesting that ability to cope with defective mitotic cells is major process for tissue development and homeostasis (Morelli, 2016).

Multiple functions of the SNARE protein Snap29 in autophagy, endocytic, and exocytic trafficking during epithelial formation in Drosophila

How autophagic degradation is linked to endosomal trafficking routes is little known. This study screened a collection of uncharacterized Drosophila mutants affecting membrane transport to identify new genes that also have a role in autophagy. A loss of function mutant was isolated in Snap29 (Synaptosomal-associated protein 29 kDa), the gene encoding the Drosophila homolog of the human protein SNAP29; and its function was characterized in vivo. Snap29 contains 2 soluble NSF attachment protein receptor (SNARE) domains and a asparagine-proline-phenylalanine (NPF motif) at its N terminus and rescue experiments indicate that both SNARE domains are required for function, whereas the NPF motif is in part dispensable. Snap29 was found to interact with SNARE proteins, localizes to multiple trafficking organelles, and is required for protein trafficking and for proper Golgi apparatus morphology. Developing tissue lacking Snap29 displays distinctive epithelial architecture defects and accumulates large amounts of autophagosomes, highlighting a major role of Snap29 in autophagy and secretion. Mutants for autophagy genes do not display epithelial architecture or secretion defects, suggesting that the these alterations of the Snap29 mutant are unlikely to be caused by the impairment of autophagy. In contrast, evidence was found of elevated levels of hop-Stat92E (hopscotch-signal transducer and activator of transcription protein at 92E) ligand, receptor, and associated signaling, which might underlie the epithelial defects. In summary, these findings support a role of Snap29 at key steps of membrane trafficking, and predict that signaling defects may contribute to the pathogenesis of cerebral dysgenesis, neuropathy, ichthyosis, and palmoplantar keratoderma (CEDNIK), a human congenital syndrome due to loss of Snap29 (Morelli, 2014).

Organ development and homeostasis require concerted regulation of membrane trafficking routes, such as those governing protein secretion and endo-lysosomal degradation, and those controlling macroautophagy (autophagy hereafter), which regulates turnover of organelles and large cytoplasmic proteins. Studies in model organisms have clearly shown that the endo-lysosomal degradation pathway is required for correct organ development, due to its ability to promote degradation of signaling receptors controlling tissue growth and polarity. Such a major role of endocytosis on tissue architecture is underscored by the fact that Drosophila larval imaginal discs, a recognized model of epithelial organ development, when mutant for a number of the Endosomal Sorting Complexes Required for Transport (ESCRT) genes, display loss of polarity and overactivation of major signaling pathways, including N (Notch) and hop-Stat92E. In contrast, mutants in genes controlling autophagy often do not display loss of tissue architecture, or altered signaling phenotypes, indicating that impairment of endo-lysosomal or autophagic degradation have dramatically distinct consequences on tissue development. However, it is poorly understood which regulators of trafficking are required for formation and convergence of autophagosomes into the endosomal degradation route, and their relevance to organ development and homeostasis (Morelli, 2014).

In autophagy, double-membrane organelles called autophagosomes are formed by a phagophore that sequesters portions of the cell cytoplasm. Autophagosomes then fuse with lysosomes, in which the autophagosome content is degraded. Studies have shown that 2 ubiquitin-like conjugation systems are required for autophagosome formation, and a number of organelles, such as the endoplasmic reticulum (ER), mitochondria, the Golgi apparatus, endosomes, and the plasma membrane have all been suggested to supply membranes and factors for autophagosome formation. Research in yeast indicates that, once formed, the autophagosome fuses with the vacuole, the yeast lysosome, in a manner dependent on the GTPase Ypt7/Rab7, on the homotypic fusion and protein sorting (HOPS) complex, and on SNARE-mediated membrane fusion. In metazoans, fusion events between autophagosomes and endosomal compartments are more complex, entailing the formation of amphisomes, which arise from fusion of autophagosomes with the multivesicular body (MVB), a late endosomal organelle. Consistent with this difference, in Drosophila and in mammalian cells ESCRT proteins, which regulate endosomal sorting and MVB formation, and the PtdIns3P 5-kinase fab1, which control endosome function, are required for amphisome and autolysosome formation. Also, differently from yeast, when formation of late endosomes is blocked in Drosophila and mammalian cells, autophagosomes accumulate in the cytoplasm, suggesting that amphisome formation helps clearance of autophagic cargoes (Morelli, 2014).

The nature of SNARE-mediated fusion events occurring during formation and clearance of autophagosomes via the endo-lysosomal system is partly obscure. SNARE-mediated fusion involves a stereotypic set of SNARE proteins forming a 4-helix bundle composed by distinct SNARE domains named Qa-, Qb-, Qc- or R-SNARE. Usually, a Qa-SNARE-containing protein (a syntaxin, or t-SNARE) and a R-SNARE -containing protein (a VAMP protein, or v-SNARE) are carried by opposing membranes, and each provide a SNARE domain to the fusion complex. These proteins are glued together by Qb- and Qc- containing proteins, providing the remaining 2 SNARE domains. The Qb- and Qc-SNAREs involved in fusion events can be contributed by members of the SNAP protein family, with SNAP25 and SNAP23 being the most extensively studied. However, metazoan genomes also contain SNAP29, which, unlike other SNAP family members, contains a N-terminal NPF (asparagine-proline-phenylalanine) motif that binds endocytic adaptors, such as EDH1, and lacks palmitoylation sites for membrane anchoring. Consistent with this, SNAP29 resides in the cytoplasm and associates with membranes transiently. In contrast to its paralogs, SNAP29 has been much less studied and its function is unclear. In tissue culture and in in vitro studies, SNAP29 has been suggested to interact with multiple Qa-SNAREs such as syntaxins, and to associate with a number of intracellular organelles to promote-as well as inhibit-membrane fusion. Using depletion approaches, it has been shown that SNAP29 and its homolog in C. elegans and zebrafish regulates trafficking between several organelles, and that it is required for integrity of various intracellular compartments. Finally, in Drosophila and human cells, the SNAREs STX17/syntaxin 17 (Syx17) and vesicle-associated membrane protein 7 (VAMP7/Vamp7) have been very recently reported to act with SNAP29/Snap29 in fusion of autophagosomes to lysosomes (Takats, 2013; Itakura, 2012). Homozygous nonsense mutations leading to truncations of the human SNAP29 protein cause CEDNIK syndrome, a rare inherited congenital condition affecting skin and nervous system development and homeostasis, and resulting in short life span.32,33 Despite the evidence above, how SNAP29 functions and how its loss results in acquisition of CEDNIK traits is currently unclear (Morelli, 2014).

This study used Drosophila imaginal discs to identify novel regulators of membrane trafficking that might have a role in autophagy, and to assess the importance of identified genes for epithelial organ development. With this strategy, the first Drosophila null mutant in Snap29 (also referred to as CG11173/usnp) was identified. Snap29 mutant imaginal discs present impairment of a late step of autophagy. In addition, it was found that Snap29 exerts an inhibitory role in membrane fusion at the apical membrane. In fact, Snap29 mutant tissue secretes autophagosomes in the apical lumen and presents excess of receptors on the plasma membrane. These defects correlate with disruption of the epithelial organization of imaginal discs and with a dramatic alteration in developmental signaling. Taken together, these data highlight a novel point of contact between trafficking and autophagy routes that is critical for organ development and might advance understanding of the CEDNIK pathogenesis (Morelli, 2014).

The identity of SNARE proteins regulating the subsequent steps of fusion required for autophagosome formation and maturation into autolysosomes is a long-standing question, on which significant progress has been reported recently. The SNAREs STX12/STX13, Ykt6, Vamp7, and Sec22 have been recently proposed to be required for autophagosome formation in yeast, Drosophila and mammals. In yeast, the SNAREs Vam3, Vam7, and Vti1 have all been suggested to control fusion of autophagosomes with vacuoles. While Vam3 and Vam7 have no clear homologs in metazoan animals, the mammalian SNAREs VAMP7, VAMP8, and VTI1B are all suggested to be involved in autophagosomal fusion events. The Qa-SNARE protein STX17 is required for membrane fusion at 2 distinct steps of autophagy: Early autophagosome formation and fusion of autophagosomes with lysosomes to form autolysosomes. An association of Syx17 with Snap29 and the R-SNARE protein Vamp7 to form a fusion complex specific for late step of autophagy has been also very recently reported in the Drosophila fat tissue (Takats, 2013). Additionally, a certain degree of accumulation of autophagosomes has been observed in C. elegans depleted of Snap-29. Ultrastructural analysis showing clearly accumulation of almost exclusively fully formed autophagosomes with preserved luminal content strongly favors the model that Snap29 is required with Syx17 and Vamp7 for fusion of autophagosomes with lysosomes. Consistent with this evidence, accumulation of autophagosomes was found in Syx17 and Vamp7 mutant discs, and a genetic interaction was detected between Snap29 and Syx17, and Snap29 and Vamp7 (Morelli, 2014).

An aspect that demands further investigation is whether Snap29 acts elsewhere in the endolysosomal system. Contrasting evidence was found for this. On one end, partial colocalization was found of Snap29 with the endosomal Qa-SNARE Syx7, and Syx7 was repeatedly found in immunoprecipitations. In addition, in uptake assays, in mutant cells the endocytic cargo N accumulates in an endosomal compartment. On the other end, such compartment is Syx7 negative. Since accumulation of N in a Syx7-positive endosomes has been reported to promote ectopic N activation, and this study has found reduced N signaling in Snap29 mutant discs, the point of N accumulation could be a postsorting compartment, such as the late endosome/MVB, or the lysosome. Despite this, no MVB accumulation was found in Snap29 mutant discs. These data are in sharp contrast with the accumulation of MVBs, but not of autophagosomes, that is observed in epithelial tissue mutant for vacuolar H+-ATPase (V-ATPase) subunit genes. Interestingly, in addition to enabling lysosomal functioning, V-ATPase have been proposed to play a role in membrane fusion and in autophagy. However, in addition to lack of accumulation of MVBs, very little sign is found of acid-induced degradation in the autophagosomes accumulated in Snap29 mutant cells. Thus, the comparison between the EM findings in Snap29 and V-ATPase mutants suggests that Snap29 functions upstream of V-ATPase in autophagy and argues against a role of V-ATPase in autophagosome formation or fusion to lysosomes (Morelli, 2014).

Traits were observed in Snap29 mutant cells that could be the result of excess or inappropriate membrane fusion events, rather than of reduced fusion. These are: the large amount of membranes forming the accumulated autophagosomes; the presence in these of folded, multilamellar membranes; the secretion of autophagosomes extracellularly. It is unlikely that these events are an indirect result from the need of mutant cells to get rid of autophagic cargoes. In fact, no autophagosome secretion or excess membrane was found around autophagosomes in Syx17 and Vamp7 mutant discs. Alternatively, excess autophagosome membrane and secretion could both arise from failure to inhibit excess vesicle fusion. Inhibitory SNAREs have been postulated to occur naturally to control Golgi stack fusion patterns, while bacteria encode inhibitory SNAREs containing 2 SNARE domains, that can act with STX7 and VAMP8 (the homologs of Drosophila Syx7 and Vamp7) to inhibit secretion of lysosomes in mammalian cells. Interestingly, negative regulation of fusion by SNAP29 at the plasma membrane has been observed in rat neurons. A direct role of Snap29 in inhibition of membrane fusion at the plasma membrane during secretion could account also for the elevated N and dome levels on the surface of mutant cells. Consistent with this possibility, it was found that Snap29 interacts with Syx1A and Syx4, plasma membrane syntaxins and can localize to the plasma membrane upon overexpression. Of note, unconventional secretion routes involving autophagy regulators have been recently described, suggesting a scenario in which the autophagy and secretion functions of Snap29 could be connected to a putative negative role in fusion. The nature of Snap29 function in fusion events, and its involvement in unconventional secretion routes are currently under investigation (Morelli, 2014).

Despite the large body of evidence on SNAP29, the pathogenesis of CEDNIK, a human congenital syndrome due to loss of Snap29, is obscure. Genetic analysis reveals that the Drosophila Snap29B6 mutant behaves as a strong loss of function and expresses a nonfunctional Snap29 protein, a similar situation to that reported for CEDNIK. Considering the absence of mouse mutants for Snap29, the findings in Drosophila could provide an initial framework to understand the pathogenesis of CEDNIK, which starts during fetal development and affects epithelial organs. In this regard, it was observed that the in vivo effect of lack of Snap29 during development in Drosophila is also epithelial tissue disorganization. This phenotype is unlikely to be due to impaired autophagy. In fact, genes specifically acting during autophagy, such as Atg13, Syx17, and Vamp7 were found to be dispensable for eye disc development. In addition, Atg7 appears dispensable for skin barrier formation in mice and flies. This evidence predicts that impairment of autophagy does not cause the developmental alterations associated to CEDNIK at least in the skin, which have been fairly well characterized. It is well possible that impaired autophagy plays a role in the unexplored neuronal traits of CEDNIK, considering that autophagy is a major process preventing neurodegeneration (Morelli, 2014)

Which of the nonautophagy defects associated to lack of Snap29 could then be relevant to skin pathogenesis in CEDNIK? Could it be the defect highlighted by N accumulation in late endosomal and lysosomal compartments in an uptake experiment? This hypothesis is not favored. In fact, this study did not detect ectopic N activation, which is a feature of mutants of ESCRT genes controlling endosomal sorting. Such difference suggests that in Snap29 mutant cells, the pool of N accumulating intracellularly has been subjected to MVB sorting and resides in the late endosomal and lysosomal lumen. Considering also that loss of genes that control post MVB sorting events generally does not perturb disc epithelium development, the defect highlighted by intracellular N accumulation in Snap29 mutant cells is per se unlikely to contribute to the developmental phenotypes of Snap29 mutant organs (Morelli, 2014).

Excluding routes that converge on the lysosomes, a further possibility is that the epithelial defects are due to alteration of secretory trafficking. Increased N presence at the plasma membrane, coupled with decreased N activation, could be relevant, since loss of N signaling is known to lead to epithelial alterations in skin. Alternatively, excess hop-Stat92E signaling could be important. In this case, excess signaling could directly originate from increased levels of active dome on the surface of Snap29 mutant cells. This scenario is consistent with the fact that Drosophila mutants preventing cargo internalization, such as those disrupting clathrin, display increased level of cargoes at the plasma membrane and possess elevated hop-Stat92E signaling and reduced N signaling. Underscoring a possible problem at the plasma membrane, expression of Socs36E, a negative regulator of hop-Stat92E signaling reported to act also by enhancing endosomal degradation of Dome, rescues part of the epithelial defects of Snap29 mutant discs. Alternatively, elevated Hop-Stat92E signaling could be a secondary effect of epithelial architecture or trafficking alterations. Detailed analysis of secretion and of signaling activity in CEDNIK samples will reveal whether alteration of these processes play a role in the pathogenesis of the syndrome (Morelli, 2014).

In summary, this study clarifies the function of Snap29 in membrane trafficking and its consequences for epithelial tissue development, which might prove relevant for human health (Morelli, 2014).

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).

Molecular mechanisms of developmentally programmed crinophagy in Drosophila

At the onset of metamorphosis, Drosophila salivary gland cells undergo a burst of glue granule secretion to attach the forming pupa to a solid surface. This study shows that excess granules evading exocytosis are degraded via direct fusion with lysosomes, a secretory granule-specific autophagic process known as crinophagy. This study found that the tethering complex HOPS (homotypic fusion and protein sorting); the small GTPases Rab2, Rab7, and its effector, PLEKHM1; and a SNAP receptor complex consisting of Syntaxin 13, Snap29, and Vamp7 are all required for the fusion of secretory granules with lysosomes. Proper glue degradation within lysosomes also requires the Uvrag-containing Vps34 lipid kinase complex and the v-ATPase proton pump, whereas Atg genes involved in macroautophagy are dispensable for crinophagy. This work establishes the molecular mechanism of developmentally programmed crinophagy in Drosophila and paves the way for analyzing this process in metazoans (Csizmadia, 2017).

Autophagosomal Syntaxin17-dependent lysosomal degradation maintains neuronal function in Drosophila

During autophagy, phagophores capture portions of cytoplasm and form double-membrane autophagosomes to deliver cargo for lysosomal degradation. How autophagosomes gain competence to fuse with late endosomes and lysosomes is not known. This paper show that Syntaxin17 is recruited to the outer membrane of autophagosomes to mediate fusion through its interactions with ubisnap (SNAP-29) and VAMP7 in Drosophila melanogaster. Loss of these genes results in accumulation of autophagosomes and a block of autolysosomal degradation during basal, starvation-induced, and developmental autophagy. Viable Syntaxin17 mutant adults show large-scale accumulation of autophagosomes in neurons, severe locomotion defects, and premature death. These mutant phenotypes cannot be rescued by neuron-specific inhibition of caspases, suggesting that caspase activation and cell death do not play a major role in brain dysfunction. These findings reveal the molecular mechanism underlying autophagosomal fusion events and show that lysosomal degradation and recycling of sequestered autophagosome content is crucial to maintain proper functioning of the nervous system (Takats, 2013).

The hairpin-type tail-anchored SNARE syntaxin 17 targets to autophagosomes for fusion with endosomes/lysosomes

The lysosome is a degradative organelle, and its fusion with other organelles is strictly regulated. In contrast to fusion with the late endosome, the mechanisms underlying autophagosome-lysosome fusion remain unknown. This study has identified syntaxin 17 (Stx17) as the autophagosomal SNARE required for fusion with the endosome/lysosome. Stx17 localizes to the outer membrane of completed autophagosomes but not to the isolation membrane (unclosed intermediate structures); for this reason, the lysosome does not fuse with the isolation membrane. Stx17 interacts with SNAP-29 and the endosomal/lysosomal SNARE VAMP8. Depletion of Stx17 causes accumulation of autophagosomes without degradation. Stx17 has a unique C-terminal hairpin structure mediated by two tandem transmembrane domains containing glycine zipper-like motifs, which is essential for its association with the autophagosomal membrane. These findings reveal a mechanism by which the SNARE protein is available to the completed autophagosome (Itakura, 2012).


REFERENCES

Search PubMed for articles about Drosophila

Csizmadia, T., Lorincz, P., Hegedus, K., Szeplaki, S., Low, P. and Juhasz, G. (2017). Molecular mechanisms of developmentally programmed crinophagy in Drosophila. J Cell Biol 217(1):361-374. PubMed ID: 29066608

Itakura, E., Kishi-Itakura, C. and Mizushima, N. (2012). The hairpin-type tail-anchored SNARE syntaxin 17 targets to autophagosomes for fusion with endosomes/lysosomes. Cell 151(6): 1256-1269. PubMed ID: 23217709

Morelli, E., Ginefra, P., Mastrodonato, V., Beznoussenko, G. V., Rusten, T. E., Bilder, D., Stenmark, H., Mironov, A. A. and Vaccari, T. (2014). Multiple functions of the SNARE protein Snap29 in autophagy, endocytic, and exocytic trafficking during epithelial formation in Drosophila. Autophagy 10(12): 2251-2268. PubMed ID: 25551675

Morelli, E., Mastrodonato, V., Beznoussenko, G. V., Mironov, A. A., Tognon, E. and Vaccari, T. (2016). An essential step of kinetochore formation controlled by the SNARE protein Snap29. EMBO J [Epub ahead of print]. PubMed ID: 27647876

Rotem-Yehudar, R., Galperin, E. and Horowitz, M. (2001). Association of insulin-like growth factor 1 receptor with EHD1 and SNAP29. J Biol Chem 276(35): 33054-33060. PubMed ID: 11423532

Steegmaier, M., Yang, B., Yoo, J. S., Huang, B., Shen, M., Yu, S., Luo, Y. and Scheller, R. H. (1998). Three novel proteins of the syntaxin/SNAP-25 family. J Biol Chem 273(51): 34171-34179. PubMed ID: 9852078

Su, Q., Mochida, S., Tian, J. H., Mehta, R. and Sheng, Z. H. (2001). SNAP-29: a general SNARE protein that inhibits SNARE disassembly and is implicated in synaptic transmission. Proc Natl Acad Sci U S A 98(24): 14038-14043. PubMed ID: 11707603

Takats, S., Nagy, P., Varga, A., Pircs, K., Karpati, M., Varga, K., Kovacs, A. L., Hegedus, K. and Juhasz, G. (2013). Autophagosomal Syntaxin17-dependent lysosomal degradation maintains neuronal function in Drosophila. J Cell Biol 201(4): 531-539. PubMed ID: 23671310

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

Venkei, Z., Przewloka, M. R., Ladak, Y., Albadri, S., Sossick, A., Juhasz, G., Novak, B. and Glover, D. M. (2012). Spatiotemporal dynamics of Spc105 regulates the assembly of the Drosophila kinetochore. Open Biol 2(2): 110032. PubMed ID: 22645658


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date revised: 6 August 2018

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