InteractiveFly: GeneBrief

yata: Biological Overview | References

yata mutants of Drosophila melanogaster exhibit phenotypes including progressive brain shrinkage, developmental abnormalities and shortened lifespan, whereas in mammals, null mutations of the yata ortholog Scyl1 result in motor neuron degeneration. yata mutation also causes defects in the anterograde intracellular trafficking of a subset of proteins including APPL, which is the Drosophila ortholog of mammalian APP, a causative molecule in Alzheimer's disease. SCYL1 binds and regulates the function of coat protein complex I (COPI) in secretory vesicles. This study reveals a role for the Drosophila YATA protein in the proper localization of COPI. Immunohistochemical analyses performed using confocal microscopy and structured illumination microscopy showed that YATA colocalizes with COPI and GM130, a cis-Golgi marker. Analyses using transgenically expressed YATA with a modified N-terminal sequence revealed that the N-terminal portion of YATA is required for the proper subcellular localization of YATA. Analysis using transgenically expressed YATA proteins in which the C-terminal sequence was modified revealed a function for the C-terminal portion of YATA in the subcellular localization of COPI. Notably, when YATA was mislocalized, it also caused the mislocalization of COPI, indicating that YATA plays a role in directing COPI to the proper subcellular site. Moreover, when both YATA and COPI were mislocalized, the staining pattern of GM130 revealed Golgi with abnormal elongated shapes. Thus, these in vivo data indicate that YATA plays a role in the proper subcellular localization of COPI (Saito, 2021).

In eukaryotic cells, transmembrane proteins and secreted proteins are synthesized in the rough endoplasmic reticulum (ER) and then transported to their own destinations by intracellular vesicular trafficking. Transport vesicles are surrounded by coat proteins, whose type varies among distinct cellular locations. These coat proteins enable the efficient formation of transport carriers and incorporation of specific cargos into the vesicles. In the early steps of vesicular trafficking between the ER and the Golgi, vesicles are coated by coat protein complex I (COPI) or II (COPII) (Miller, 2013; Arakel, 2018; Bethune, 2018). COPI-coated vesicles transport proteins retrogradely from the Golgi to the ER, whereas COPII-coated vesicles transport proteins anterogradely from the ER to the Golgi. Proper regulation of vesicular trafficking between the ER and the Golgi is considered to be important in the retrieval of ER- and Golgi-resident proteins as well as appropriate quality control of synthesized proteins. Impairment of the early steps of vesicular trafficking has been suggested to cause some human genetic disorders (Saito, 2021).

A Drosophila yata mutant was previously identified that showed phenotypes in the compound eye, wing and brain. Homozygotes of the null allele of the yata gene, yata^KE2.1, show various phenotypes such as morphological abnormalities in the compound eye and wing, progressive reduction of brain volume and shortened lifespan. Electron microscopic examination of the internal morphology of the compound eye revealed that the yata mutation enhanced the formation of an abnormal cellular structure that formed as a bleb-like cellular protrusion and contained membranous organelles that had the appearance of lysosomes, autophagosomes and late endosomes (Arimoto, 2020). The yata gene has been suggested to be ubiquitously expressed. It encodes a protein that has no transmembrane domains and has a catalytic domain of a protein kinase. The kinase domain of YATA is predicted to be catalytically inactive because some of the amino acid residues that are essential for catalytic activity are not conserved. The yata mutant was originally identified as a locus that genetically interacted with the null allele of the Appl gene. The Appl gene encodes the orthologous protein of mammalian APP, which is a causative molecule of Alzheimer's disease. Double null mutants of yata and Appl showed exacerbated phenotypes in brain volume reduction and shortened lifespan. APPL proteins are synthesized in neuronal cell bodies and then transported to synaptic terminals by means of vesicular trafficking. Immunohistochemical analysis using an anti-APPL antibody revealed the aberrant accumulation of APPL in neuronal cell bodies in the pupal brain in yata mutants. This accumulation of APPL overlapped with a marker of the ER. In a Drosophila Alzheimer's disease model in which human mutant APP was ectopically expressed in larval motor neurons, RNAi-mediated knockdown of yata resulted in partial suppression of the anterograde transport of APP to synapses and of phenotypes caused by APP such as abnormal morphology of neuromuscular synapses and abnormal electrophysiological properties of neuromuscular synapses (Furotani, 2018). In addition to APPL, synaptic transport of another synaptic protein, Fasciclin II, which is a synaptic homophilic cell adhesion molecule orthologous to mammalian NCAM, is reduced in yata mutants. However, synaptic transport of Synaptotagmin I, which is a membrane-bound protein localized on synaptic vesicles, was not affected in the yata mutants, suggesting that yata mutation affects only a subset of proteins that are transported by vesicular trafficking (Saito, 2021).

yata orthologs occur in a variety of eukaryotes including yeast, plants, nematodes and mammals. Null mutation of the murine yata ortholog, Scyl1, causes motor neuron degeneration (Schmidt, 2007). Because neural-specific, but not skeletal muscle-specific, deletion of Scyl1 causes motor dysfunction, Scyl1 is thought to function autonomously in the nervous system (Pelletier, 2012). Because deletion of Scyl1 also caused the mislocalization and accumulation of the TAR DNA-binding protein of 43 kDa (TDP-43), mice with Scyl1 deletion are thought to share features with human neurodegenerative diseases, including amyotrophic lateral sclerosis. Mutation of human Scyl1 has been identified as a cause of a genetic disease that results in liver failure, peripheral neuropathy, cerebellar atrophy and ataxia (Schmidt, 2015; Lenz, 2018; Shohet, 2019). A previous mass spectrometry-based screen identified βCOP, a subunit of COPI, as the binding partner of SCYL1 (Burman, 2008). SCYL1 directly binds with COPI and colocalizes with COPI in the ER–Golgi intermediate compartment (ERGIC) and the cis-Golgi, which are the sites where buds of COPI-coated vesicles are formed. In cultured cells, RNAi-mediated knockdown of Scyl1 resulted in the inhibition of COPI-mediated retrograde trafficking of the KDEL receptor protein from the Golgi to the ER. Further study also identified class II Arfs, which are GTPases that are involved in the formation of COPI-coated vesicles, as another binding partner of SCYL1 (Hamlin, 2014). SCYL1 has been suggested to function as a scaffold protein for components of the COPI coat. In cultured cells, overexpression or knockdown of Scyl1 resulted in abnormal morphology of the Golgi and ERGIC, possibly due to loss of the scaffolding function of SCYL1 for COPI (Burman, 2010; Hamlin, 2014). In patients with a genetic disease caused by a mutation in Scyl1, enlargement of the Golgi has been observed in fibroblasts (Saito, 2021).

This study used immunostaining with anti-YATA and other marker antibodies to examine the subcellular localization of YATA in the Drosophila pupal brain. Observations obtained using confocal microscopy and structured illumination microscopy (SIM) revealed colocalization among YATA, COPI and a cis-Golgi marker. Thie further analysis using transgenically expressed YATA indicate that it has an in vivo role in the proper subcellular localization of COPI (Saito, 2021).

This study examined the subcellular localization of the YATA protein by immunostaining with an anti-YATA antibody and observation using confocal microscopy. Anti-YATA signals were observed to have a punctate pattern in the cytoplasm surrounding nuclei. These punctate signals colocalized with the signals for GM130, a marker of the cis-Golgi, and for COPI. Further analysis using SIM revealed that COPI and YATA were localized in a subset of regions within the GM130 punctae. YATA and COPI partially colocalized. These data suggest that the cis-Golgi, which is labeled by anti-GM130 antibody, contains several distinct subregions where YATA and COPI are localized. Subregions where GM130 colocalized with both YATA and COPI may be sites for the assembly and budding of COPI-coated secretory vesicles traveling from the Golgi to the ER. Consistent with this interpretation, when the expression of Arf4-GFP was induced, its localization showed mainly punctate signals that overlapped with the localization of YATA. A previous study has shown that Arf4-GFP is localized in punctae that seem to be the sites of formation of COPI-coated vesicles in Drosophila embryos, although it has not been proven that localization of Arf4-GFP completely overlaps with that of the endogenous Arf4 protein. On the other hand, when YFP-KDEL expression was induced, it was found to be localized in the regions that surrounded the nuclei and in some regions near the nuclei. The localization of YATA partially overlapped the YFP-KDEL signal, but the patterns and shapes presented by the signals were different. The C-terminal KDEL sequence is the ER retention motif, which is recognized by the KDEL receptor and functions in the retrieval of ER-resident proteins from the Golgi. In Drosophila neurons, staining with an anti-KDEL antibody and the localization of RFP-KDEL and Lysozyme-GFP-KDEL all reveal localization in the regions that surround the nuclei and in some regions near the nuclei. In addition, a previous immunoelectron microscopic study showed that a secreted protein, Hikaru genki, is localized in the region surrounding the nucleus in Drosophila neurons. These observations suggest that the regions found to be labeled by YFP-KDEL represent the ER. When the expression of Sar1-HA was induced, Sar1-HA showed a relatively strong punctate pattern of localization and a relatively weak pattern of perinuclear localization that seemed to correspond to the ER. A previous study has shown that Sar1-GFP is localized in the perinuclear region and that it also appears as relatively strongly stained punctae in Drosophila S2 cells. Costaining with dSec16 suggested that the relatively strongly stained punctae represent the ER exit sites at which COPII-coated vesicles are formed. Because the localization of Sar1-HA resembles that of Sar1-GFP, the punctate signals that arise from Sar1-HA seem to represent the ER exit sites, although it has not been proven that localization of Sar1-HA completely overlaps with that of endogenous Sar1 protein. YATA colocalized with the punctate Sar1-HA signal. Notably, the punctate signals arising from anti-YATA antibody labeling may not represent the entire areas of YATA localization. When YATAwt was overexpressed with no epitope tag, relatively weak staining was also observed in the perinuclear regions of the cytoplasm in addition to the relatively strong punctate signals that were found to colocalize with COPI and the cis-Golgi marker. Therefore, its COPI-related function may not be the only molecular function of YATA. In addition, the site of assembly of COPI-coated vesicles on the cis-Golgi and the site of assembly of COPII-coated vesicles on the ER may be spatially close to each other (Saito, 2021).

This study also assessed whether overexpression of YATA affects the localization of COPI. The results suggest that ectopic overexpression of YATAwt with no epitope tag increased the localization of COPI. This is consistent with the hypothesis that YATA directs COPI to the subregion of the cis-Golgi where COPI-coated vesicles are assembled, because an increased amount of YATA may be capable of directing an increased amount of COPI to this specific subregion of the cis-Golgi. However, in cultured cells, overexpression of SCYL1 conjugated with GFP caused an expanded morphology of the cis-Golgi, as revealed by labeling with anti-GM130 antibody (Hamlin, 2014). These differences may be due to species-specific differences, differences between cultured cells and living animals, an effect of the conjugated GFP, or differences in gene dosage (Saito, 2021).

Previous studies have shown that SCYL1 binds COPI at its C-terminal amino acid sequence (RKLD-COO-), which resembles the KKXX-COO- motif that functions as a COPI-binding motif in ER-resident proteins. Because the Drosophila YATA protein has an AKKL-COO- sequence at its C-terminus, whether mutating the C-terminal sequence of the YATA protein would influence the effect of YATA on COPI localization was tested. While overexpression of YATAwt increased the localization of COPI, the induction of YATAwt-HA, which has a 3×HA tag sequence at its C-terminus, did not affect COPI. These data suggest that the C-terminal sequence of YATA is required for its effect on COPI. The effects of HA-YATAwt and HA-YATAAAAL expression, both of which have a 3×HA tag at their N-terminus, were also tested. It was unexpectedly found that these N-terminally tagged YATA proteins did not exactly colocalize with the cis-Golgi marker but instead showed diffuse localization in the cytoplasm. These data suggest that the N-terminal sequence of YATA is required for its proper subcellular localization. Although the reason for the importance of the N-terminal sequence is unknown, mislocalization of N-terminally tagged YATA enabled examination of the effect of mistakenly localized YATA on the localization of COPI. The data show that the expression of mislocalized HA-YATAwt caused the mislocalization of COPI, while the expression of mislocalized HA-YATAAAAL did not. These data suggest that YATA functions to direct COPI to the proper subcellular site and that the C-terminal sequence of YATA is required for this function. The data regarding the localization of a cis-Golgi marker show that the Golgi exhibited an abnormal elongated shape when HA-YATAwt was expressed but that this effect was not observed when HA-YATAAAAL was expressed. These data suggest that the expression of mislocalized YATA protein that retains its original C-terminal sequence caused the cis-Golgi to acquire abnormal morphology. These observations are consistent with previous results showing that the overexpression or knockdown of Scyl1 resulted in an expanded morphology of the Golgi in cultured cells (Burman, 2010; Hamlin, 2014) and that the Golgi was enlarged in fibroblasts of patients with a genetic disease caused by mutation of Scyl1 (Schmidt, 2015; Saito, 2021 and references therein).

The data reveal a function of YATA, namely its involvement in the regulation of subcellular localization of COPI, and provide a basis for further in vivo genetic explorations of the mechanisms and physiological importance of COPI-mediated vesicular trafficking in Drosophila. However, the relevance of the COPI-regulating function of YATA to the phenotypes observed in yata null mutants is still unclear. yata mutants show impaired anterograde trafficking of a subset of proteins including APPL and Fasciclin II, although Synaptotagmin I trafficking is not affected (Sone, 2009; Furotani, 2018). In addition, the aberrant accumulation of Sec23, a component of COPII, was observed in yata mutants. One possibility is that the impairment of COPI-mediated retrograde trafficking from the Golgi to the ER disrupts the quality control system and metabolism of proteins synthesized in the ER, as previously suggested, because the retrograde trafficking system is necessary to recover some of the proteins that play roles in the ER and are transported from the ER to the Golgi. Alternatively, YATA could have another function in addition to the regulation of the subcellular localization of COPI in which it could affect the anterograde trafficking of some proteins. The important question of why the trafficking of only a subset of proteins is affected in yata mutants also remains unresolved. The fact that null mutants of yata are not lethal suggests that there are other molecules whose functions overlap with that of yata. Paralogs of yata that exist in the Drosophila genome and belong to the family of genes that encode SCYL family pseudokinases are candidates for such molecules. Further detailed analysis of the phenotypes observed in yata mutants and their relevance to the formation of COPI-coated vesicles and the regulation of intracellular vesicular trafficking will be necessary in the future (Saito, 2021).

Analysis of a cellular structure observed in the compound eyes of Drosophila white; yata mutants and white mutants

Previous work has identified the Drosophila yata mutant, which showed phenotypes including progressive vacuolization of the white-colored compound eye, progressive shrinkage of the brain and a shortened lifespan. The yata gene was shown to be involved in controlling intracellular trafficking of the APPL protein, which is an orthologue of APP that is a causative molecule of Alzheimer's disease. This study examined the phenotype of the compound eye of the yata mutant using electron microscopy and confocal microscopy. Abnormal cellular structures that seemed to originate from bleb-like structures and contained vesicles and organelles, such as multivesicular bodies and autophagosomes, were observed in aged white; yata mutants and aged white mutants. These structures were not observed in newly eclosed flies, and the presence of the structures was suppressed in flies grown under constant dark conditions after eclosion. The structures were not observed in newly eclosed red-eyed yata mutants or wild-type flies but were observed in very aged red-eyed wild-type flies. Thus, these data suggest that the observed structures are formed as a result of changes associated with exposure to light after eclosion in white mutants, white; yata mutants and aged flies (Arimoto, 2020).

In this study, the phenotypes of the compound eyes of yata mutants were observed in fine detail using electron microscopy. The observations led to the identification of abnormal cellular structures located in the vicinity of rhabdomeres in aged day 15 and day 29 white; yata mutants. In tangential sections, rhabdomeres were connected to the cytoplasm of the cells from which they were derived, and a different side of these rhabdomeres was often also attached to the identified structures. Although the identified structures were surrounded by a plasma membrane, they were often attached to rhabdomeres. The identified structures were usually located near or opposite the central cavity. In horizontal sections, the identified structures were often found in between rod-like rhabdomeres, and were found to be both connected to and separated from rhabdomeres. They were also found lateral to rhabdomeres and embedded in the cytoplasm of photoreceptor neurons, and were also occasionally observed to cause twisting of the rhabdomeres. Because the identified structures seem to adhere to rhabdomeres, there may be adhesive mechanisms between the identified structures and rhabdomeres. In addition, in some electron micrographs, the identified structures were observed to be bleb-like structures that extended from the cytoplasm of photoreceptor neurons from locations near the adherens junction. These observations suggest that the identified structures originate as bleb-like structures from the cytoplasm of photoreceptor neurons and may migrate into the location of rhabdomeres or the cytoplasm of photoreceptor neurons. The identified structures were filled with vesicles, vacuoles and membranous organelles including multivesicular bodies that appeared to be late endosomes, structures with double membranes and electron-dense structures. Analysis using confocal microscopy revealed the accumulation of anti-Atg8a and anti-Rab7 signals near the rhabdomeres in the day 15 white; yata mutants. Anti-Rab7 antibody has been shown to label late endosomes. Anti-Atg8a antibody has previously been shown to specifically detect the Drosophila Atg8a protein in immunostaining of the fat body of third instar larvae. Although previous studies showed that anti-Atg8 antibody can label aggregates of ectopically overexpressed Atg8 protein or Atg8 protein incorporated into protein aggregates in cultured cells, the anti-Atg8a signal observed in the white; yata mutants seems to represent the accumulation of autophagosomes, as it is consistent with the observation that the identified structures contain structures with double membranes, as revealed by electron microscopy. Thus, these data suggest that the identified structures contained autophagosomes and late endosomes. Moreover, electron-dense structures observed in the identified structures appeared to be similar to lysosomes. These findings suggest that the identified structures may contain organelles involved in protein degradation pathways, such as the autophagy-lysosome system (Arimoto, 2020).

In addition to their presence in day 15 and day 29 white; yata mutants, the identified structures were found in day 29 white mutants. However, the identified structures were not found in day 1 white; yata or white mutants. These structures may be similar to 'multivesicular body-like bodies' previously observed in aged day 21 white mutants. These 'multivesicular body-like bodies' were found to occasionally form a peninsula-like structure that extended from the cytoplasm of a photoreceptor neuron near the adherens junction, and this observation is consistent with the bleb-like form of the identified structures that were observed. The identified structures were not found in red-eyed yata mutant and wild-type flies on days 1 and 29. Formation of the identified structures was completely suppressed in day 29 white mutants reared under constant dark conditions after eclosion. These findings suggest that formation of the structure in white mutants is caused by changes in photoreceptor neurons associated with excessive exposure to light after eclosion because pigment granules were absent in the white mutants. In addition, the data showed that the identified structures also formed in very aged day 71 red-eyed wild-type flies. These changes may be a component of the changes associated with ageing in the compound eyes of flies (Arimoto, 2020).

Significantly more identified structures were observed in the day 29 white; yata mutants than in the day 29 white mutants reared under 12-h light/12-h dark conditions. The identified structures were observed in the day 15 white; yata mutants, but not in the day 15 white mutants. Therefore, the yata mutation may enhance formation of the identified structures, although whether the yata mutation enhances formation of the identified structures in flies with a red-eyed background is unclear. The photoreceptor neurons of yata mutants, at least those in the white mutant background, may be more vulnerable to changes associated with exposure to light. In addition, formation of the identified structures was not completely suppressed in day 29 white; yata mutants reared under constant dark conditions after eclosion, although no identified structures were observed in day 29 white mutants reared under constant dark conditions after eclosion. Therefore, the yata mutation may enhance the mechanisms that lead to formation of the identified structures, even in flies reared under constant dark conditions after eclosion. The loss of such yata-dependent mechanisms may cause formation of the identified structures in the background of a null mutation of white or in flies in which pigment granules have been lost. Diverse phenotypes in addition to a white eye colour have been described in white mutants, suggesting a wide range of molecular functions for the white gene. Although why white mutation enhanced formation of the identified structures in white; yata mutants reared under constant dark conditions after eclosion is unclear, this effect may be related to the fact that pigment granules are lysosome-like organelles and that several genes involved in the biogenesis of pigment granules also play a role in lysosome trafficking. Notably, giant multivesicular structures with diameters greater than 3 μm have been observed in the pigment cells of mutants of the deep orange gene, which encodes an endosomal protein required for normal eye colour (Arimoto, 2020).

The data suggest that the identified structures are more frequently observed in ommatidia at the R8 level than in those at the R7 level regardless of genotype or age. These observations suggest that the identified structures form more frequently in a proximal position in the retina than in a distal position in the retina. Although the reason for this location-dependent differential vulnerability is unknown, it may be related to a previous observation that retinal degeneration processes occurred more rapidly in proximal positions remote from the nucleus than in distal positions in rdgA mutants. Therefore, the difference in the frequency of the identified structures between the ommatidia at the R8 and R7 levels may be related to the susceptibility of photoreceptor neurons to degenerative changes (Arimoto, 2020).

Previous studies suggested that yata is involved in regulating the intracellular trafficking of a subset of proteins including APPL and Fasciclin II (Furotani, 2018; Sone, 2009). However, it has also been suggested that yata does not control the localization of all membrane-bound and secreted proteins transported via vesicular trafficking because the localization of Synaptotagmin was not affected by yata mutation. In addition, yata may also control the trafficking of some unidentified proteins. Therefore, the loss of yata may enhance formation of the identified structures by impairing the localization of APPL, Fasciclin II or other unidentified target proteins or an unidentified molecular function of yata (Arimoto, 2020).

In conclusion, this study identified a cellular structure that contains vesicles, autophagosomes and multivesicular bodies in the compound eyes of aged white; yata mutants, aged white mutants and very aged wild-type flies. The data suggest that these structures are formed as a result of changes associated with exposure to light after eclosion in white mutants, white; yata mutants and aged flies. Further exploration of the mechanisms of formation of the identified structures may clarify their physiological significance (Arimoto, 2020).

The dimeric Golgi protein Gorab binds to Sas6 as a monomer to mediate centriole duplication

Gorab shows many of the properties typical of golgins, a family of tentacle-like proteins that protrude from the Golgi membranes to capture a variety of target vesicles. Redundancy between golgins in their ability to bind target vesicles could act as a functional safeguard and might explain why loss-of-function gorab mutants display no obvious Golgi phenotype, contrasting to the Golgi defects shown by the C-terminally tagged Gorab molecule. Gorab is similar to other golgins, which also associate with the Golgi membranes through their C-terminal parts in interactions that require Rab family member proteins to interact with the C-terminal part of the golgin dimer. The N-terminal parts of the golgins interact with their vesicle targets. Human GORAB's N-terminal part interacts with Scyl1 to promote the formation of COPI vesicles at the trans-Golgi (Witkos, 2019). However, its precise role in the transport of COPI vesicles is not clear, particularly why loss of human GORAB affects Golgi functions in just bone and skin when COPI function is required in multiple tissues. Drosophila Gorab also co-purifies and physically interacts with both Yata, counterpart of Scyl1, and COPI vesicle components, and its importance for transport of COPI vesicles in Drosophila is similarly unclear (Fatalska, 2021).

Suppression of the synaptic localization of a subset of proteins including APP partially ameliorates phenotypes of the Drosophila Alzheimer's disease model

APP (amyloid precursor protein), the causative molecule of Alzheimer's disease, is synthesized in neuronal cell bodies and subsequently transported to synapses. It has been shown that the yata gene is required for the synaptic transport of the APP orthologue in Drosophila melanogaster. This study examined the effect of a reduction in yata expression in the Drosophila Alzheimer's disease model, in which expression of human mutant APP was induced. The synaptic localization of APP and other synaptic proteins was differentially inhibited by yata knockdown and null mutation. Expression of APP resulted in abnormal synaptic morphology and the premature death of animals. These phenotypes were partially but significantly rescued by yata knockdown, whereas yata knockdown itself caused no abnormality. Moreover, synaptic transmission accuracy was impaired in this model, and this phenotype was improved by yata knockdown. Thus, these data suggested that the phenotypes caused by APP can be partially prevented by inhibition of the synaptic localization of a subset of synaptic proteins including APP (Furotani, 2018).

This study examined whether the inhibition of the synaptic localization of APP affects the phenotypes of the Drosophila model of Alzheimer's disease. For this purpose, the yata gene. The yata gene is required for the intracellular transport of the APPL protein, which is the Drosophila orthologue of mammalian APP. This study induced the expression of the human Swedish mutant APP in larval motor neurons. Knockdown and null mutation of yata resulted in decreased synaptic localization of APP. In this study, immunostaining was performed using the anti-APP antibody MAb 4G8 that recognizes amino acid residues 17–24 of the amyloid β region. Therefore, the observed localization was the expression for full-length APP or the processed fragment that contains the amyloid β region. Notably, yata mutation resulted in the differential suppression of the synaptic localization of a subset of proteins including APP without uniformly affecting all of the synaptic molecules. The data showed that synaptic localization of APP was impaired whereas localization of Fasciclin II was not significantly affected by yata knockdown. Localization of Synaptotagmin and Cysteine string protein was also not significantly affected even in yata null mutants. Such selectivity is a desired property of a tool that therapeutically targets APP. On the other hand, localization of Fasciclin II was significantly decreased in the yata null mutants. yata has a mammalian orthologue, SCYL1. Although both yata and SCYL1 are suggested to be involved in the trafficking of the coated secretory vesicles, the data suggested that the impact of yata loss-of-function is differential among proteins that are transported by vesicular trafficking. The data suggested that APP and Fasciclin II are affected by yata mutation. As a possibility, specific secretory vesicles that contain a subset of proteins such as APP and Fasciclin II as cargos are relatively severely affected by yata mutation. Another molecule whose synaptic localization was found to be affected in yata mutants was the Bruchpilot protein, which is a cytoplasmic protein and is a component of the electron-dense T-bar structures of the presynaptic active zone. This finding was unexpected, because yata/SCYL1 is suggested to play a role in the trafficking of secretory vesicles. Therefore, synaptic dysfunction caused by impaired synaptic development may affect the assembly of presynaptic components such as Bruchpilot. Alternatively, yata may also be involved in the expression or trafficking of synaptic cytoplasmic proteins (Furotani, 2018).

Knockdown of yata partially ameliorated abnormalities in the Alzheimer's disease model, such as increased number of satellite boutons, lethality during development, lethality in the first 10 days after eclosion and impaired synaptic transmission accuracy. In addition, heterozygous introduction of the yata null allele also partially rescued the developmental lethality and lethality within 10 days after eclosion, although the effect was weaker and developmental lethality was not rescued in males. yata knockdown and heterozygosity of the yata null allele themselves caused no apparent abnormalities. The only phenotype observed to be caused by yata knockdown was the elevated variance in the amplitudes of eEJPs. While homozygotes of the yata null allele show phenotypes including developmental abnormalities, progressive brain volume reduction and shortened lifespan, heterozygotes of the yata null allele are viable and fertile and show no apparent abnormalities like many other recessive mutations. The reason why the knockdown of yata causes no apparent abnormalities may be because the effect of knockdown is partial and decreased expression of yata is still sufficient for most of molecular functions of yata. On the other hand, most of the observed rescuing effects were only partial. This finding may possibly be because the synaptic localization of induced APP is still sufficient for the expression of some phenotypes, although the synaptic localization was decreased. Alternatively, synapse-independent mechanisms controlled by induced APP may exist (Furotani, 2018).

Lifespan data showed that the lethality caused by the expression of APP is restricted to the developmental stage from the embryo to the the first 10 days of the adult stage after eclosion. The animals that survived past this stage showed an almost normal lifespan, suggesting that the cause of death is a defect in neural development. Because lethality can be suppressed by yata knockdown and possibly by the decreased synaptic localization of a subset of proteins including APP, this finding suggests that the cause of death is a defect in synapses. In addition, electrophysiological analysis revealed that the expression of APP resulted in impaired synaptic transmission accuracy, because expression of APP caused a significantly elevated variance in the amplitudes of eEJPs, while the amplitudes and frequencies of mEJPs were not significantly affected. The phenotype of the variance of eEJPs was suppressed by yata knockdown and possibly by the decreased synaptic localization of a subset of proteins including APP. Such instability of synaptic transmission may be a result of impaired synaptic development. Notably, knockdown of yata also caused a impairment in the accuracy of synaptic transmission similar to the phenotype caused by the expression of APP, potentially reflecting the physiological function of yata in synaptic physiology. While synaptic localization of ectopically induced APP is decreased by yata knockdown, synaptic localization of other endogenous synaptic proteins may also be affected, and this might contribute to the elevated variance in eEJPs by yata knockdown. Knockdown of yata caused elevated eEJP variance but did not cause premature lethality of animals. These data are against the possibility that the instability of synaptic transmission directly causes the death of animals. The identity of synapses that contribute significantly to the death of animals is also unknown. In this model, the excessive formation of satellite boutons was observed in the neuromuscular synapses on the body wall muscles of larvae. This means that the expression of APP resulted in the loss of proper control of synapse formation. Such a loss of control may cause fatal results in the synapses that are essential for the survival of animals (Furotani, 2018).

In this study, the expression of human Swedish mutant APP associated with familial Alzheimer's disease was induced. Among the phenotypes observed in this study, excessive formation of satellite boutons is known to be caused by the overexpression of human wild-type APP and Drosophila Appl. Therefore, these phenotypes are suggested to be caused by the elevated gene dosage of Appl/APP. It is necessary to examine the contribution of APP mutation to other phenotypes including observed electrophysiological phenotypes, especially because it has been previously shown that the overexpression of Drosophila Appl caused different electrophysiological phenotypes including a decrease in eEJP amplitude, an increase in mEJP amplitude and a decrease in quantal content, although experimental conditions including the selection of Gal4 were different (Furotani, 2018).

Synaptic loss is observed in patients with Alzheimer's disease. On the other hand, previous studies have shown that mammalian APP-family genes are involved in synaptic development. Double knock-out mice of several combinations of APP family genes show embryonic lethality, possibly caused by defects in synaptic morphology and function. Although the APP protein is transported and localized in synapses, whether the role of APP in synaptic morphology and function is attributed to the function of the APP protein localized in the synapses is still unclear. In this study, the data suggested that the suppression of synaptic localization of a subset of proteins including APP partially rescued the synaptic phenotypes caused by APP, suggesting the importance of the function of APP localized in synapses. On the other hand, while Alzheimer's disease is a late-onset disease, this model and double APP knock-out mice show developmental defects in synapses. In fact, loss-of-function mutants of the Drosophila Appl gene show not only defects in synaptic development but also late-onset shortened lifespan. The shortened lifespan of the Appl mutants may reflect the physiological function of Appl in the maintenance of the nervous system against aging. Alternatively, developmental defects may also cause late-onset phenotypes. It remains to be elucidated if the molecular function of Appl in synapses is related to the late-onset premature death of Appl mutant flies, although the relevance of the physiological functions of APP in the pathogenesis of Alzheimer's disease is still unclear and the current model did not show the late-onset premature death of animals (Furotani, 2018).

Because Drosophila yata mutants and SCYL1 null mutant mice share neurodegeneration phenotypes, and the human SCYL1 gene has been identified as a causative gene of a genetic disease causing peripheral neuropathy and cerebellar atrophy, the molecular function of these orthologues seems to be evolutionarily conserved. The synaptic pathology of Alzheimer's disease may be able to be modified if one could control the synaptic localization of APP. Although the mammalian orthologue of yata, SCYL1, is a candidate target molecule to affect the synaptic localization of APP, complete ablation of both Drosophila yata and mammalian SCYL1 result in fatal phenotypes including neurodegeneration. Moreover, knockdown of yata itself causes impaired synaptic transmission accuracy. Therefore, it is necessary to examine if there is a way to control the synaptic localization of APP without fatal side-effects, if it is possible to control the functional expression of SCYL1 strictly and if SCYL1 can be used as a target molecule in a therapeutic approach for the treatment of Alzheimer's disease (Furotani, 2018).

Loss of yata, a novel gene regulating the subcellular localization of APPL, induces deterioration of neural tissues and lifespan shortening

The subcellular localization of membrane and secreted proteins is finely and dynamically regulated through intracellular vesicular trafficking for permitting various biological processes. Drosophila Amyloid precursor protein like (APPL) and Hikaru genki (HIG) are examples of proteins that show differential subcellular localization among several developmental stages. During the study of the localization mechanisms of APPL and HIG, a novel mutant was isolated of the gene, CG1973, which was named yata. This molecule interacted genetically with Appl and is structurally similar to mouse NTKL/SCYL1, whose mutation was reported to cause neurodegeneration. yata null mutants showed phenotypes that included developmental abnormalities, progressive eye vacuolization, brain volume reduction, and lifespan shortening. Exogenous expression of Appl or hig in neurons partially rescued the mutant phenotypes of yata. Conversely, the phenotypes were exacerbated in double null mutants for yata and Appl. The subcellular localization of endogenous APPL and exogenously pulse-induced APPL tagged with FLAG was examined by immunostaining the pupal brain and larval motor neurons in yata mutants. These data revealed that yata mutants showed impaired subcellular localization of APPL. Finally, yata mutant pupal brains occasionally showed aberrant accumulation of Sec23p, a component of the COPII coat of secretory vesicles traveling from the endoplasmic reticulum (ER) to the Golgi. Thus this study identified a novel gene, yata, which is essential for the normal development and survival of tissues. Loss of yata resulted in the progressive deterioration of the nervous system and premature lethality. The genetic data showed a functional relationship between yata and Appl. As a candidate mechanism of the abnormalities, it was found that yata regulates the subcellular localization of APPL and possibly other proteins (Sone, 2009).

Variant in SCYL1 gene causes aberrant splicing in a family with cerebellar ataxia, recurrent episodes of liver failure, and growth retardation

This study describe two members of one family who presented with recurrent episodes of hepatic failure, cerebellar ataxia, peripheral neuropathy, and short stature. Liver transplantation was considered. Whole-exome sequencing (Trio) revealed a synonymous variant in exon 4 of SCYL1:c.459C>T p. (Gly153Gly), which did not appear to affect the protein sequence. Computational prediction analysis suggested that this modification could alter the SCYL1 mRNA splicing processing to create a premature termination codon. The SCYL1 mRNAs in the patient's lymphocytes were analyzed and aberrant splicing was found. Molecular analysis of family members identified the parents as heterozygous recessive carriers and the proband as well as an affected aunt as homozygous. Evidently, harmless synonymous variants in the SCYL1 gene can damage gene splicing and hence the expression. It was confirmed that the pathogenicity of this variant in the SCYL1 gene was associated with spinocerebellar ataxia, autosomal recessive 21 (SCAR21). Other reported cases (accept one) of liver failure found in the SCYL1 variants resolved during childhood, therefore orthotropic liver transplantation was no longer appropriate (Shohet, 2019).

SCYL1 variants cause a syndrome with low gamma-glutamyl-transferase cholestasis, acute liver failure, and neurodegeneration (CALFAN)

Biallelic mutations in SCYL1 were recently identified as causing a syndromal disorder characterized by peripheral neuropathy, cerebellar atrophy, ataxia, and recurrent episodes of liver failure. The occurrence of SCYL1 deficiency among patients with previously undetermined infantile cholestasis or acute liver failure has not been studied; furthermore, little is known regarding the hepatic phenotype. This study aimed to identify patients with SCYL1 variants within an exome-sequencing study of individuals with infantile cholestasis or acute liver failure of unknown etiology. Deep clinical and biochemical phenotyping plus analysis of liver biopsies and functional studies on fibroblasts were performed. Seven patients from five families with biallelic SCYL1 variants were identified. The main clinical phenotype was recurrent low gamma-glutamyl-transferase (GGT) cholestasis or acute liver failure with onset in infancy and a variable neurological phenotype of later onset (CALFAN syndrome). Liver crises were triggered by febrile infections and were transient, but fibrosis developed. Functional studies emphasize that SCYL1 deficiency is linked to impaired intracellular trafficking. It is concluded that SCYL1 deficiency can cause recurrent low-GGT cholestatic liver dysfunction in conjunction with a variable neurological phenotype. Like NBAS deficiency, it is a member of the emerging group of congenital disorders of intracellular trafficking causing hepatopathy (Lenz, 2018).

SCYL2 Protects CA3 Pyramidal Neurons from Excitotoxicity during Functional Maturation of the Mouse Hippocampus

Neuronal death caused by excessive excitatory signaling, excitotoxicity, plays a central role in neurodegenerative disorders. The mechanisms regulating this process, however, are still incompletely understood. This study shows that the coated vesicle-associated kinase SCYL2/CVAK104 plays a critical role for the normal functioning of the nervous system and for suppressing excitotoxicity in the developing hippocampus. Targeted disruption of Scyl2 in mice caused perinatal lethality in the vast majority of newborn mice and severe sensory-motor deficits in mice that survived to adulthood. Consistent with a neurogenic origin of these phenotypes, neuron-specific deletion of Scyl2 also caused perinatal lethality in the majority of newborn mice and severe neurological defects in adult mice. The neurological deficits in these mice were associated with the degeneration of several neuronal populations, most notably CA3 pyramidal neurons of the hippocampus, which were analyzed in more detail. The loss of CA3 neurons occurred during the functional maturation of the hippocampus and was the result of a BAX-dependent apoptotic process. Excessive excitatory signaling was present at the onset of degeneration, and inhibition of excitatory signaling prevented the degeneration of CA3 neurons. Biochemical fractionation reveals that Scyl2-deficient mice have an altered composition of excitatory receptors at synapses. These findings demonstrate an essential role for SCYL2 in regulating neuronal function and survival and suggest a role for SCYL2 in regulating excitatory signaling in the developing brain (Gingras, 2015).

Scyl1 scaffolds class II Arfs to specific subcomplexes of coatomer through the gamma-COP appendage domain

Coatomer (COPI)-coated vesicles mediate membrane trafficking in the early secretory pathway. There are at least three subclasses of COPI coats and two classes of Arf GTPases that couple COPI coat proteins to membranes. Whether mechanisms exist to link specific Arfs to specific COPI subcomplexes is unknown. This study demonstrates that Scy1-like protein 1 (Scyl1), a member of the Scy1-like family of catalytically inactive protein kinases, oligomerizes through centrally located HEAT repeats and uses a C-terminal RKXX-COO(-) motif to interact directly with the appendage domain of coatomer subunit gamma-2 (also known as COPG2 or gamma2-COP). Through a distinct site, Scyl1 interacts selectively with class II Arfs, notably Arf4, thus linking class II Arfs to gamma2-bearing COPI subcomplexes. Therefore, Scyl1 functions as a scaffold for key components of COPI coats, and disruption of the scaffolding function of Scyl1 causes tubulation of the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) and the cis-Golgi, similar to that observed following the loss of Arf and Arf-guanine-nucleotide-exchange factor (GEF) function. These data reveal that Scyl1 is a key organizer of a subset of the COPI machinery (Hamlin, 2014).

An early onset progressive motor neuron disorder in Scyl1-deficient mice is associated with mislocalization of TDP-43

The molecular and cellular bases of motor neuron diseases (MNDs) are still poorly understood. The diseases are mostly sporadic, with ~10% of cases being familial. In most cases of familial motor neuronopathy, the disease is caused by either gain-of-adverse-effect mutations or partial loss-of-function mutations in ubiquitously expressed genes that serve essential cellular functions. This study shows that deletion of Scyl1, an evolutionarily conserved and ubiquitously expressed gene encoding the COPI-associated protein pseudokinase SCYL1, causes an early onset progressive MND with characteristic features of amyotrophic lateral sclerosis (ALS). Skeletal muscles of Scyl1(-/-) mice displayed neurogenic atrophy, fiber type switching, and disuse atrophy. Peripheral nerves showed axonal degeneration. Loss of lower motor neurons (LMNs) and large-caliber axons was conspicuous in Scyl1(-/-) animals. Signs of neuroinflammation were seen throughout the CNS, most notably in the ventral horn of the spinal cord. Neural-specific, but not skeletal muscle-specific, deletion of Scyl1 was sufficient to cause motor dysfunction, indicating that SCYL1 acts in a neural cell-autonomous manner to prevent LMN degeneration and motor functions. Remarkably, deletion of Scyl1 resulted in the mislocalization and accumulation of TDP-43 (TAR DNA-binding protein of 43 kDa) and ubiquilin 2 into cytoplasmic inclusions within LMNs, features characteristic of most familial and sporadic forms of ALS. Together, these results identify SCYL1 as a key regulator of motor neuron survival, and Scyl1(-/-) mice share pathological features with many human neurodegenerative conditions (Pelletier, 2012).

Search PubMed for articles about Drosophila Yata

Arakel, E. C. and Schwappach, B. (2018). Formation of COPI-coated vesicles at a glance. J Cell Sci 131(5). PubMed ID: 29535154

Arimoto, E., Kawashima, Y., Choi, T., Unagami, M., Akiyama, S., Tomizawa, M., Yano, H., Suzuki, E. and Sone, M. (2020). Analysis of a cellular structure observed in the compound eyes of Drosophila white; yata mutants and white mutants. Biol Open 9(1). PubMed ID: 31862863

Bethune, J. and Wieland, F. T. (2018). Assembly of COPI and COPII Vesicular Coat Proteins on Membranes. Annu Rev Biophys 47: 63-83. PubMed ID: 29345989

Fatalska, A., Stepinac, E., Richter, M., Kovacs, L., Pietras, Z., Puchinger, M., Dong, G., Dadlez, M. and Glover, D. M. (2021). The dimeric Golgi protein Gorab binds to Sas6 as a monomer to mediate centriole duplication. Elife 10. PubMed ID: 33704067

Furotani, K., Kamimura, K., Yajima, T., Nakayama, M., Enomoto, R., Tamura, T., Okazawa, H. and Sone, M. (2018). Suppression of the synaptic localization of a subset of proteins including APP partially ameliorates phenotypes of the Drosophila Alzheimer's disease model. PLoS One 13(9): e0204048. PubMed ID: 30226901

Gingras, S., Earls, L. R., Howell, S., Smeyne, R. J., Zakharenko, S. S. and Pelletier, S. (2015). SCYL2 Protects CA3 Pyramidal Neurons from Excitotoxicity during Functional Maturation of the Mouse Hippocampus. J Neurosci 35(29): 10510-10522. PubMed ID: 26203146

Hamlin, J. N., Schroeder, L. K., Fotouhi, M., Dokainish, H., Ioannou, M. S., Girard, M., Summerfeldt, N., Melancon, P. and McPherson, P. S. (2014). Scyl1 scaffolds class II Arfs to specific subcomplexes of coatomer through the gamma-COP appendage domain. J Cell Sci 127(Pt 7): 1454-1463. PubMed ID: 24481816

Lenz, D., McClean, P., Kansu, A., Bonnen, P. E., Ranucci, G., Thiel, C., Straub, B. K., Harting, I., Alhaddad, B., Dimitrov, B., Kotzaeridou, U., Wenning, D., Iorio, R., Himes, R. W., Kuloglu, Z., Blakely, E. L., Taylor, R. W., Meitinger, T., Kolker, S., Prokisch, H., Hoffmann, G. F., Haack, T. B. and Staufner, C. (2018). SCYL1 variants cause a syndrome with low gamma-glutamyl-transferase cholestasis, acute liver failure, and neurodegeneration (CALFAN). Genet Med 20(10): 1255-1265. PubMed ID: 29419818

Miller, E. A. and Schekman, R. (2013). COPII - a flexible vesicle formation system. Curr Opin Cell Biol 25(4): 420-427. PubMed ID: 23702145

Pelletier, S., Gingras, S., Howell, S., Vogel, P. and Ihle, J. N. (2012). An early onset progressive motor neuron disorder in Scyl1-deficient mice is associated with mislocalization of TDP-43. J Neurosci 32(47): 16560-16573. PubMed ID: 23175812

Saito, M., Nakayama, M., Fujita, K., Uchida, A., Yano, H., Goto, S., Okazawa, H. and Sone, M. (2021). Role of the Drosophila YATA protein in the proper subcellular localization of COPI revealed by in vivo analysis. Genes Genet Syst. PubMed ID: 33583916

Schmidt, W. M., Kraus, C., Hoger, H., Hochmeister, S., Oberndorfer, F., Branka, M., Bingemann, S., Lassmann, H., Muller, M., Macedo-Souza, L. I., Vainzof, M., Zatz, M., Reis, A. and Bittner, R. E. (2007). Mutation in the Scyl1 gene encoding amino-terminal kinase-like protein causes a recessive form of spinocerebellar neurodegeneration. EMBO Rep 8(7): 691-697. PubMed ID: 17571074

Schmidt, W. M., Rutledge, S. L., Schule, R., Mayerhofer, B., Zuchner, S., Boltshauser, E. and Bittner, R. E. (2015). Disruptive SCYL1 Mutations Underlie a Syndrome Characterized by Recurrent Episodes of Liver Failure, Peripheral Neuropathy, Cerebellar Atrophy, and Ataxia. Am J Hum Genet 97(6): 855-861. PubMed ID: 26581903

Shohet, A., Cohen, L., Haguel, D., Mozer, Y., Shomron, N., Tzur, S., Bazak, L., Basel Salmon, L. and Krause, I. (2019). Variant in SCYL1 gene causes aberrant splicing in a family with cerebellar ataxia, recurrent episodes of liver failure, and growth retardation. Eur J Hum Genet 27(2): 263-268. PubMed ID: 30258122

Sone, M., Uchida, A., Komatsu, A., Suzuki, E., Ibuki, I., Asada, M., Shiwaku, H., Tamura, T., Hoshino, M., Okazawa, H. and Nabeshima, Y. (2009). Loss of yata, a novel gene regulating the subcellular localization of APPL, induces deterioration of neural tissues and lifespan shortening. PLoS One 4(2): e4466. PubMed ID: 19209226

date revised: 12 January 2022

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