InteractiveFly: GeneBrief

skywalker: Biological Overview | References

Gene name - skywalker

Synonyms -

Cytological map position - 38F3-38F5

Function - signaling

Keywords - a RabGAP that activates GTPase activity of the product of Rab35, neuromuscular junction, restricts the ability of synaptic vesicles to fuse into a synaptic endosomal compartment, genetically interacts with components of the ESCRT and HOPS complexes

Symbol - sky

FlyBase ID: FBgn0032901

Genetic map position - chr2L:20,871,335-20,885,976

NCBI classification - Rab-GTPase-TBC domain

Cellular location - cytoplasmic

NCBI links: EntrezGene, Nucleotide, Protein

Synaptic demise and accumulation of dysfunctional proteins are thought of as common features in neurodegeneration. However, the mechanisms by which synaptic proteins turn over remain elusive. Drosophila melanogaster was studied lacking active TBC1D24/Skywalker (Sky), a protein that in humans causes severe neurodegeneration, epilepsy, and DOOR (deafness, onychdystrophy, osteodystrophy, and mental retardation) syndrome, and identify endosome-to-lysosome trafficking as a mechanism for degradation of synaptic vesicle-associated proteins. In fly sky mutants, synaptic vesicles traveled excessively to endosomes. Using chimeric fluorescent timers, it was shown that synaptic vesicle-associated proteins were younger on average, suggesting that older proteins are more efficiently degraded. Using a genetic screen, it was found that reducing endosomal-to-lysosomal trafficking, controlled by the homotypic fusion and vacuole protein sorting (HOPS) complex, rescued the neurotransmission and neurodegeneration defects in sky mutants. Consistently, synaptic vesicle proteins were older in HOPS complex mutants, and these mutants also showed reduced neurotransmission. These findings define a mechanism in which synaptic transmission is facilitated by efficient protein turnover at lysosomes and identify a potential strategy to suppress defects arising from TBC1D24 mutations in humans (Fernandes, 2014).

Loss-of-function mutations in human TBC1D24 cause severe neurodegeneration, focal and infantile myoclonic epilepsy, malignant migrating partial seizures of infancy, intellectual disability, and DOOR (deafness, onychdystrophy, osteodystrophy, and mental retardation) syndrome. However, the molecular nature of the defects upon loss of TBC1D24 remains poorly characterized (Fernandes, 2014).

TBC1D24 encodes an evolutionarily conserved GTPase-activating protein (Uytterhoeven, 2011). In flies, Skywalker (Sky)/TBC1D24 resides at synapses and inhibits Rab35-mediated synaptic vesicle trafficking in a pathway parallel to Rab5 (Uytterhoeven, 2011). Rab5 and Rab35 promote synaptic vesicles to fuse with endosomes, and this additional trafficking step correlates with increased transmitter release and a larger pool of readily releasable vesicles (RRPs). However, it is not clear how fusion of vesicles with endosomes results in increased neurotransmitter release (Fernandes, 2014).

It was hypothesized that endosomes may serve as sorting stations for synaptic vesicle proteins, whereby dysfunctional synaptic vesicle proteins are removed from the vesicle cycle at endosomes and sent to the lysosome for degradation. When the dysfunctional vesicle proteins are removed from the vesicle cycle, synaptic vesicles would be populated by the remaining functional vesicle proteins, thus facilitating neurotransmission. In an unbiased modifier screen for sky mutant phenotypes, a homotypic fusion and vacuole protein sorting (HOPS) complex component Deep orange (Dor)/VPS18, was identified; Dor been previously described to control endosome-to-lysosomal trafficking (Rieder, 1997). The HOPS complex consists of Vps11, 16, 18, 33, 39, and 41 and binds to proteins that mediate fusion of cargo vesicles with the lysosome. Using a fluorescent synaptic vesicle-associated timer, this study shows that the HOPS complex is critical for synaptic vesicle protein turnover. Furthermore, similar to loss of TBC1D24 in humans, sky mutant flies show massive brain lesions reminiscent of neurodegeneration, which can also be rescued by the partial inhibition of Dor. This work unveils a mechanism by which endosome-to-lysosomal trafficking controls synaptic vesicle protein turnover, ensuring proper levels of neurotransmitter release as well as neuronal survival in the context Sky/TBC1D24 function (Fernandes, 2014).

Loss of sky/TBC1D24 in humans causes neurodegeneration and other neuronal defects. To identify genetic modifiers of sky, a genetic screen was performed in fruit flies. Sky mutants die in development, but flies homozygously mutant for sky only in their eyes, survive, and show defects in electroretinograms (ERGs). ERGs measure the difference in potential between the eye and the body during a light flash. Sky mutant eyes result in smaller on and off transients in ERG traces, indicating desynchronized neuronal communication. To identify dominant modifiers of the sky ERG phenotype, a previously isolated collection of 111 X-linked mutants was used. These chemically randomly induced (ethyl methanesulfonate [EMS]) mutations were isolated because they show defects in neurotransmission when homozygous in the eye, and it was therefore reasoned they would be good candidates to genetically interact with sky. Flies homozygous in the eye for sky and heterozygous for one of the 111 mutations were generate, and ERGs were recorded. Although most mutants do not affect the sky-induced ERG defect, 26 of the 111 mutants show rescue. Interestingly, three suppressor mutants, dor30, dor35, and dor36 were identified that also fail to complement one another based on lethality, suggesting they are alleles of the same gene (Fernandes, 2014).

To identify the genetic lesions in the dor30, dor35, and dor36 mutant alleles, mapping was combined with whole genome sequencing, identified a splice donor mutation was identified resulting in a protein with 56 additional amino acids and an early stop codon in the case of dor35 and nonsense mutations in dor36 and dor30. Immunoblotting revealed lower Dor protein levels in dor36 and dor30 mutants, whereas dor35 is still expressed (though is a dysfunctional protein). Thus, these three alleles appear to result in loss of Dor function. In agreement with this, the larval lethality associated with the dor alleles can be rescued by ubiquitous expression of Dor (daGAL4) (Fernandes, 2014).

Dor is an orthologue of Vps18, a member of the HOPS complex, which promotes trafficking of vesicles to lysosomes for degradation in yeast. To assess whether Dor has a similar function in flies, the conversion of the endolysosomal protease Cathepsin L from its proform into mature Cathepsin L was measured in dor mutants. Pro-Cathepsin L is delivered to the lysosome by HOPS-dependent transport, in which its propeptide is subsequently cleaved and the protease becomes active. Therefore, it would be expected that defects in HOPS-dependent transport would result in less Cathepsin L delivery to lysosomes and thus an accumulation of the propeptide in comparison to its mature form. Indeed, all dor mutants were found to show an accumulation of pro-Cathepsin L, indicating that Dor is also required for normal lysosomal trafficking in flies (Fernandes, 2014).

To assess whether flies with homozygous mutant sky2 retinae constitute a model for TBC1D24-induced neurodegeneration, histological analysis was performed. The morphology of the retina of sky2 mutant eyes in one-day-old flies is very similar to controls. However, in a 10-d period under constant light, many vacuoles accumulate. Next, retinae were examined that are homozygous for sky2 and heterozygous for the different dor alleles. Consistent with the dor-dependent rescue of ERG defects, an almost complete suppression was found of the neurodegeneration induced by loss of sky (Fernandes, 2014).

Neurodegeneration is often linked to abnormal synaptic transmission. Accordingly, sky mutants show a significant increase in neurotransmission. To determine whether loss of dor suppresses the neurotransmission defects in sky mutants, two-electrode voltage clamp was used at the larval neuromuscular junction (NMJ). The excitatory junctional current (EJC) in sky mutants is 77% larger than in controls, and removing one copy of dor suppresses this defect. This rescue is specific to loss of dor because expression of Dor (daGAL4) in sky; dor/+ mutants likewise results in much larger EJCs. These data indicate that Dor down-regulation rescues the increased neurotransmitter release in sky mutants (Fernandes, 2014).

Previous work has shown that loss of sky causes synaptic vesicles to cycle excessively to endosomes (Uytterhoeven, 2011). To assess whether loss of dor also suppresses this defect, larval fillets were incubated in FM 1-43 under stimulating conditions, resulting in the uptake of the dye into newly formed vesicles. In sky mutants, the dye concentrates in aberrant accumulations, which are positive for the endosomal marker Rab5-GFP (Uytterhoeven, 2011), indicative of increased synaptic vesicle trafficking to endosomes. Despite the observation that loss of Dor rescues the increased synaptic transmission in sky mutants, the occurrence of Rab5-positive FM 1-43 accumulations persist in dor/+; sky double mutants. Thus, reduced Dor function can rescue defects in neurotransmitter release but does not prevent vesicles from excessively cycling to endosomal compartments in sky mutants (Fernandes, 2014).

Dor is a member of the HOPS complex and to assess whether other HOPS components or proteins involved in lysosomal trafficking also suppress aspects of the sky mutant phenotype, the neuronal driver nSybGAL4 and RNAi were used to knock down vps39/CG7146 and rab7 (knockdown efficiency by quantitative RT-PCR: 36% CG7146 and 40% rab7). Similar to partial loss of Dor, RNAi to vps39 or rab7 suppresses the increased EJC amplitude in sky mutants but not the aberrant accumulations of FM 1-43. These data suggest that trafficking of vesicles to endosomes in sky mutants is not sufficient to facilitate neurotransmitter release but that efficient HOPS complex-dependent traffic to lysosomes is required as well (Fernandes, 2014).

To assess the contribution of Dor-dependent lysosomal traffic in exocytosis of synaptic vesicles, the mean amplitude of EJCs and of spontaneous mini-events (miniature EJCs [mEJCs]) were measured. Although the mean EJC amplitudes in dor mutants and controls are similar, the mEJC amplitudes in both dor35 and dor36 mutants are larger compared with controls and can be rescued by restoring the expression of Dor ubiquitously. Hence, the quantal content (EJC/mEJC) in dor mutants is reduced compared with controls, suggesting that in dor mutants, less vesicles fuse with the synaptic membrane upon nerve stimulation (Fernandes, 2014).

Larger mEJCs can originate either from larger vesicles that harbor more neurotransmitters or from increased synaptic glutamate receptor abundance. Quantification of synaptic vesicle diameter using transmission EM (TEM) of dor35 and dor36 mutant synaptic boutons does not reveal a difference in mean synaptic vesicle size compared with controls. However, dor35 and dor36 mutants display increased glutamate receptor labeling at synapses (anti-GluRIIA intensity normalized to anti-HRP). These data are therefore consistent with the hypothesis that the larger miniatures observed in dor mutants originate postsynaptically because of a larger glutamate receptor field (Fernandes, 2014).

The presynaptic contribution of Dor to synaptic vesicle release was further tested by presynaptically expressing Dor at NMJs of dor mutants (vGlutGAL4). This condition does not rescue the larger mEJCs that were observed in dor mutants. However, compared with dor mutants, larger EJCs and higher quantal content were found in dor mutants expressing Dor presynaptically. These data indicate that Dor regulates quantal content in a cell-autonomous manner (Fernandes, 2014).

To further test the conclusion that fewer vesicles fuse upon stimulation in dor mutants, FM 1-43 dye labeling of NMJ nerve terminals was used. Vesicle fusion elicits reformation of new vesicles from the plasma membrane that can be labeled by FM 1-43, providing a measure of presynaptic vesicle cycling. dor mutants were restimulated in the presence of FM 1-43, and the distribution and amount of dye uptake was measured. FM 1-43 distributes very similar to controls, but the dor mutants internalize less dye. Indicating specificity, expression of wild-type Dor using vGlutGAL4 or daGAL4 at least partially rescues the defect. Hence, loss of dor at presynaptic terminals results in reduced vesicle cycling during stimulation (Fernandes, 2014).

Next, the nature of the presynaptic defects observed in dor mutants was probed by evaluating synaptic vesicle number, the presence of endocytic intermediates, and the number of vesicles tethered at presynaptic release sites. None of these parameters is affected in dor mutants, suggesting Dor does not majorly influence vesicle reformation and tethering. The size of the RRP was measured. The cumulative released number of quanta was measured during a short high frequency stimulation train (60 Hz and 600 ms), and the trend line was back extrapolated between 400 and 600 ms. The RRP was ~30% smaller in dor mutants compared with controls, suggesting the defect in quantal content observed in dor mutants may be attributed, at least in part, to a smaller RRP. Previous work with mutants that affect the function of the lysosome also show reduced neurotransmitter release, but it is not known whether they too affect the RRP (Fernandes, 2014).

The data indicate that reduced lysosomal trafficking, as observed in dor mutants, results in a smaller RRP and that the increased neurotransmitter release in sky mutants is dependent on normal levels of endolysosomal trafficking. Based on these data, it is hypothesized that lysosomal trafficking is needed for synaptic vesicle protein turnover: In this model, defective endolysosomal traffic in dor mutants causes the buildup of older vesicle proteins. It is surmised that the longer a protein is in use (i.e.,the older it is), the higher the probability of damage, and it is proposed that the buildup of old proteins results in a less efficient release apparatus. To test this model, a synaptic vesicle-associated protein (neuronal synaptobrevin [nSyb]) was fused to a fluorescent timer (FT) protein that changes from blue to red emission over time, allowing assessment of protein-pool age. Newly synthesized nSyb is blue, whereas older nSyb is red, and thus the ratio of red to blue fluorescence provides a measure of protein pool age (Fernandes, 2014).

Flies expressing FT-nSyb under control of vGlutGAL4 were generated and red and blue fluorescence was observed at synapses. At NMJs, FT-nSyb labeling is visible in a typical ring-like pattern that colocalizes with the synaptic vesicle marker cysteine string protein (CSP). Furthermore, when the synaptic vesicles are allowed to fuse with the membrane, but their endocytosis is blocked using a temperature-sensitive dynamin (shits1), the FT-nSyb redistributes more to the presynaptic membrane. These data indicate that FT-nSyb properly localizes to and associates with synaptic vesicles (Fernandes, 2014).

Whether expression of the FT-nSyb affects synaptic function was assessed. FT-nSyb-expressing flies survive, can fly, and do not show obvious behavioral defects. In addition, synaptic vesicle cycling in these animals as measured by FM 1-43 dye uptake upon stimulation is similar to controls. Thus, expression of the FT-nSyb fusion protein does not overtly affect synaptic function (Fernandes, 2014).

Next, the FT-nSyb was used to assess synaptic vesicle protein turnover. First, whether the FT-nSyb is a protein that can be efficiently degraded was assessed by fusing it to ubiquitin (Ub), forcing the FT-nSyb to be targeted for degradation. Under identical expression conditions, the synaptic labeling of Ub-FT-nSyb is much lower than that of FT-nSyb, and red fluorescence is almost undetectable. These results indicate fast and efficient degradation of the ubiquitinated protein. As a further control, the FT-nSyb was expressed in animals expressing constitutive active Rab35 in which traffic of synaptic vesicles to endosomes is enhanced, and old proteins are expected to be degraded more efficiently (Uytterhoeven, 2011). A lower red over blue ratio was found, consistent with a younger synaptic vesicle protein pool in Rab35CA-expressing animals. FT-nSyb was then expressed in dor35 and dor36 mutants and in sky1/2 mutants, hypothesizing that older proteins will dwell longer in dor mutants and less long in sky mutants. In agreement, a lower ratio of red-to-blue fluorescence was found in sky mutants and a higher ratio in dor mutants. Furthermore, this defect in sky mutants is reversed when one copy of dor is removed. Hence, the data are consistent with the idea that Dor-mediated endolysosomal trafficking positively regulates synaptic vesicle protein turnover (Fernandes, 2014).

This work has establish a fly model of Sky/TBC1D24-induced neurodegeneration and has found that reduced function of Dor, a HOPS component, is sufficient to suppress this neurodegeneration. These data indicate that the synaptic vesicle protein pool in the sky mutants is on average younger compared with controls, suggesting excessive degradation of older proteins, thereby promoting synaptic vesicle protein pool rejuvenation. This degradation of older proteins in sky mutants is dependent on Dor-mediated endolysosomal trafficking. Indeed, reducing Dor activity results in older, and likely partly dysfunctional proteins, to be kept in the vesicle cycle. Although not directly shown, it is surmised that newly synthesized proteins are on average more functional than aged proteins, and these aged proteins may engage in inefficient complexes that dampen the efficacy of neurotransmitter release. Excessive degradation of these older proteins in sky mutants indeed correlates with increased neurotransmitter release and with neurodegeneration, both features that are suppressed when partially blocking endolysosomal traffic in the sky mutants (Fernandes, 2014).

TBC1D24-TLDc-related epilepsy exercise-induced dystonia: rescue by antioxidants in a disease model

Genetic mutations in TBC1D24 have been associated with multiple phenotypes, with epilepsy being the main clinical manifestation. The TBC1D24 protein consists of the unique association of a Tre2/Bub2/Cdc16 (TBC) domain and a TBC/lysin motif domain/catalytic (TLDc) domain. More than 50 missense and loss-of-function mutations have been described and are spread over the entire protein. Through whole genome/exome sequencing compound heterozygous mutations, R360H and G501R, were identified within the TLDc domain, in an index family> with a Rolandic epilepsy exercise-induced dystonia phenotype. A 20-year long clinical follow-up revealed that epilepsy was self-limited in all three affected patients, but exercise-induced dystonia persisted into adulthood in two. Furthermore,three additional sporadic paediatric patients were identified with a remarkably similar phenotype, two of whom had compound heterozygous mutations consisting of an in-frame deletion I81_K84 and an A500V mutation, and the third carried T182M and G511R missense mutations, overall revealing that all six patients harbour a missense mutation in the subdomain of TLDc between residues 500 and 511. The crystal structure of the conserved Drosophila TLDc domain was solved. This allowed prediction of destabilizing effects of the G501R and G511R mutations and, to a lesser degree, of R360H and potentially A500V. Next, the functional consequences of a strong and a weak TLDc mutation (TBC1D24G501R and TBC1D24R360H) was characterized using Drosophila, where TBC1D24/Skywalker regulates synaptic vesicle trafficking. In a Drosophila model neuronally expressing human TBC1D24, this study demonstrated that the TBC1D24G501R TLDc mutation causes activity-induced locomotion and synaptic vesicle trafficking defects, while TBC1D24R360H is benign. The neuronal phenotypes of the TBC1D24G501R mutation are consistent with exacerbated oxidative stress sensitivity, which is rescued by treating TBC1D24G501R mutant animals with antioxidants N-acetylcysteine amide or alpha-tocopherol as indicated by restored synaptic vesicle trafficking levels and sustained behavioural activity. These data thus show that mutations in the TLDc domain of TBC1D24 cause Rolandic-type focal motor epilepsy and exercise-induced dystonia. The humanized TBC1D24G501R fly model exhibits sustained activity and vesicle transport defects. It is proposed that the TBC1D24/Sky TLDc domain is a reactive oxygen species sensor mediating synaptic vesicle trafficking rates that, when dysfunctional, causes a movement disorder in patients and flies. The TLDc and TBC domain mutations' response to antioxidant treatment observed in the animal model suggests a potential for combining antioxidant-based therapeutic approaches to TBC1D24-associated disorders with previously described lipid-altering strategies for TBC domain mutations (Luthy, 2019).

Skywalker-TBC1D24 has a lipid-binding pocket mutated in epilepsy and required for synaptic function

Mutations in TBC1D24 cause severe epilepsy and DOORS syndrome, but the molecular mechanisms underlying these pathologies are unresolved. The crystal structure of the TBC domain of the Drosophila ortholog Skywalker was solved, revealing an unanticipated cationic pocket conserved among TBC1D24 homologs. Cocrystallization and biochemistry showed that this pocket binds phosphoinositides phosphorylated at the 4 and 5 positions. The most prevalent patient mutations affect the phosphoinositide-binding pocket and inhibit lipid binding. Using in vivo photobleaching of Skywalker-GFP mutants, including pathogenic mutants, this study showed that membrane binding via this pocket restricts Skywalker diffusion in presynaptic terminals. Additionally, the pathogenic mutations cause severe neurological defects in flies, including impaired synaptic-vesicle trafficking and seizures, and these defects are reversed by genetically increasing synaptic PI(4,5)P2 concentrations through synaptojanin mutations. Hence, this study discovered that a TBC domain affected by clinical mutations directly binds phosphoinositides through a cationic pocket and that phosphoinositide binding is critical for presynaptic function (Fischer, 2016).

Loss of skywalker reveals synaptic endosomes as sorting stations for synaptic vesicle proteins

Exchange of proteins at sorting endosomes is not only critical to numerous signaling pathways but also to receptor-mediated signaling and to pathogen entry into cells; however, how this process is regulated in synaptic vesicle cycling remains unexplored. This work presents evidence that loss of function of a single neuronally expressed GTPase activating protein (GAP), Skywalker (Sky) facilitates endosomal trafficking of synaptic vesicles at Drosophila neuromuscular junction boutons, chiefly by controlling Rab35 GTPase activity. Analyses of genetic interactions with the ESCRT machinery as well as chimeric ubiquitinated synaptic vesicle proteins indicate that endosomal trafficking facilitates the replacement of dysfunctional synaptic vesicle components. Consequently, sky mutants harbor a larger readily releasable pool of synaptic vesicles and show a dramatic increase in basal neurotransmitter release. Thus, the trafficking of vesicles via endosomes uncovered using sky mutants provides an elegant mechanism by which neurons may regulate synaptic vesicle rejuvenation and neurotransmitter release (Uytterhoeven, 2011).

Synaptic vesicles recycle locally at the synapse, and this study now provides genetic evidence that the synapse holds the capacity to regulate the sorting of synaptic vesicle proteins at 2xFYVE and Rab5 positive endosomes. 2xFYVE-GFP positive endosomes involved in signaling pathways exist at nerve endings. Likewise, membrane invaginations and endosomal-like cisternae form as a result of bulk membrane uptake during intense nerve stimulation, and in various endocytic mutants. However, the current data indicate that endosomes that accumulate in sky mutants are fundamentally different from these 'endocytic cisternae.' First, endocytic cisternae are not enriched for the endosomal markers 2xFYVE and Rab5. Second, endosomal structures in sky mutants do not appear to directly form at the plasma membrane. Third, endocytic cisternae may directly fuse with the membrane, however, based on mEJC and TEM analyses, sky induced endosomes appear to function as an intermediate station. Note that FM 1-43 dye can enter and leave endosomes in sky mutants, suggesting small synaptic vesicles can form at these structures. Together with time-course experiments in sky mutants, endosomes specifically accumulate upon stimulation and dissipate during rest. Therefore, the data suggest that endosomes in sky mutants constitute an intermediate step in the synaptic vesicle cycle. Such a compartment may also be operational in wild-type synapses because a 2xFYVE-GFP endosomal compartment can be largely depleted from synaptic vesicles upon endocytic blockade and because synaptic vesicles harbor proteins that are also commonly found on endosomes (Uytterhoeven, 2011).

Rab GTPases are involved in numerous intracellular trafficking events. In particular in synaptic vesicle traffic, Rab3, and its close isoform Rab27 have been implicated in vesicle tethering and/or priming, while Rab5 has been also implicated in endocytosis. However, an involvement of Rab proteins in other aspects of the synaptic vesicle cycle, including recycling, remains enigmatic. This systematic analysis of CA Rabs now implicates several Rabs in synaptic recycling. First, consistent with previous results, the data suggest that Rab5 mediates transport to synaptic endosomes. Second, Rab7CA and Rab11CA appear to inhibit new vesicle formation, likely by controlling post endosome trafficking. Finally, this study found that Rab23CA and Rab35CA mediate transport to or retention of synaptic vesicles at sub-boutonic structures. Neither of these Rabs had yet been implicated in synaptic vesicle traffic and hence, these in vivo studies identified different Rab proteins involved in aspects of the synaptic vesicle cycle (Uytterhoeven, 2011).

The expression of Rab5CA and Rab53CA results in increased trafficking of vesicles to -or retention of vesicles at- sub-boutonic structures, and unlike expression of Rab23CA, Rab5CA and Rab53CA also result in a facilitation of neurotransmitter release similar to sky mutants. Rab35 has been found to localize to the plasma membrane and that study shows enrichment at NMJ boutons close to the membrane as well. Rab35 has been implicated in endosomal, clathrin-dependent traffic, phagocytic membrane uptake in non-neuronal cells and actin filament assembly during Drosophila bristle development. Given these roles, Rab35 is ideally posed to also play a role in the endosomal traffic of synaptic vesicles, and the in vitro and in vivo genetic interaction studies indicate Sky to be a Rab35 GAP in synaptic vesicle trafficking. Although Rab5 mediates endosomal traffic in different cell types, The data indicate that Sky is not a GAP for Rab5 in vivo at the synapse. While this study does not exclude that Sky activates the GTPase activity of alternative Rabs in different contexts, the results suggest a Sky-Rab35 partnership that restricts endosomal trafficking of synaptic vesicles (Uytterhoeven, 2011).

In most cell types the ESCRT complex mediates sorting of ubiquitinated proteins into multivesicular bodies; however, such a function has not been characterized for synaptic vesicle components. This study provides evidence that the ESCRT complex mediates synaptic vesicle protein sorting in sky mutants where synaptic vesicles cycle excessively via endosomes. In addition, more efficient clearance of an artificially ubiquitinated synaptic vesicle protein, Ub-nSybHA, was found in sky mutants, and this effect is ESCRT dependent. Combined, the data indicate endosomal sorting to control the quality of proteins in theeth. Further underscoring this notion, in humans, mutations in the Sky orthologue TBC1D24 are causative of focal epilepsy (Uytterhoeven, 2011).

The sorting mechanism uncovered by loss of Sky function or upon expression of Rab53CA may address the use-dependent decline in protein- or lipid-function at synapses, and the continuous need to rejuvenate the synaptic vesicle protein pool. In addition, it may also constitute a mechanism to remove inappropriately endocytosed membrane components from the vesicle pool. The trafficking pathway inhibited by Sky may also yield the ability of synapses to adapt the functional properties of their synaptic vesicles, thus controlling the nature or abundance of proteins involved in vesicle fusion and neuronal signaling (Uytterhoeven, 2011).


Search PubMed for articles about Drosophila Skywalker

Fernandes, A. C., Uytterhoeven, V., Kuenen, S., Wang, Y. C., Slabbaert, J. R., Swerts, J., Kasprowicz, J., Aerts, S. and Verstreken, P. (2014). Reduced synaptic vesicle protein degradation at lysosomes curbs TBC1D24/sky-induced neurodegeneration. J Cell Biol 207: 453-462. PubMed ID: 25422373

Fischer, B., Luthy, K., Paesmans, J., De Koninck, C., Maes, I., Swerts, J., Kuenen, S., Uytterhoeven, V., Verstreken, P. and Versees, W. (2016). Skywalker-TBC1D24 has a lipid-binding pocket mutated in epilepsy and required for synaptic function. Nat Struct Mol Biol 23(11): 965-973. PubMed ID: 27669036

Luthy, K., Mei, D., Fischer, B., De Fusco, M., Swerts, J., Paesmans, J., Parrini, E., Lubarr, N., Meijer, I. A., Mackenzie, K. M., Lee, W. T., Cittaro, D., Aridon, P., Schoovaerts, N., Versees, W., Verstreken, P., Casari, G. and Guerrini, R. (2019). TBC1D24-TLDc-related epilepsy exercise-induced dystonia: rescue by antioxidants in a disease model. Brain 142(8): 2319-2335. PubMed ID: 31257402

Rieder, S. E. and Emr, S. D. (1997). A novel RING finger protein complex essential for a late step in protein transport to the yeast vacuole. Mol Biol Cell 8(11): 2307-2327. PubMed ID: 9362071

Uytterhoeven, V., Kuenen, S., Kasprowicz, J., Miskiewicz, K. and Verstreken, P. (2011). Loss of skywalker reveals synaptic endosomes as sorting stations for synaptic vesicle proteins. Cell 145(1): 117-132. PubMed ID: 21458671

Biological Overview

date revised: 2 December 2019

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