InteractiveFly: GeneBrief

snazarus: Biological Overview | References

Gene name - snazarus

Synonyms -

Cytological map position - 7C2-7C3

Function - sorting nexin, transmembrane protein

Keywords - fat body - adipocyte protein - sorting nexin - phospholipid-binding protein - associates with peripheral lipid droplets and regulates lipid droplet homeostasis at endoplasmic reticulum-plasma membrane contact sites - controls lifespan

Symbol - snz

FlyBase ID: FBgn0029976

Genetic map position - chrX:7,912,089-7,917,440

NCBI classification - PX_SNX25: The phosphoinositide binding Phox Homology domain of Sorting Nexin 25

Cellular location - ER transmembrane and associated with lipid droplets

NCBI link: EntrezGene, Nucleotide, Protein
snz orthologs: Biolitmine

Adipocytes store nutrients as lipid droplets (LDs), but how they organize their LD stores to balance lipid uptake, storage, and mobilization remains poorly understood. Using Drosophila fat body (FB) adipocytes, this study characterized spatially distinct LD populations that are maintained by different lipid pools. Peripheral LDs (pLDs) were identified that make close contact with the plasma membrane (PM) and are maintained by lipophorin-dependent lipid trafficking. pLDs are distinct from larger cytoplasmic medial LDs (mLDs), which are maintained by FASN1-dependent de novo lipogenesis. Sorting nexin CG or Snazarus (Snz) associates with pLDs and regulates LD homeostasis at ER-PM contact sites. Loss of Snz perturbs pLD organization, whereas Snz over-expression drives LD expansion, triacylglyceride production, starvation resistance, and lifespan extension through a Desaturase 1-dependent pathway. It is proposed that Drosophila adipocytes maintain spatially distinct LD populations, and Snz is identified as a regulator of LD organization and inter-organelle crosstalk (Ugrankar, 2019).

Life presents energetic and metabolic challenges, and metazoans have developed specialized nutrient-storing organs to maintain energy homeostasis and buffer against the ever-changing availability of dietary nutrients. Drosophila melanogaster is a key model organism to study energy homeostasis as many aspects of mammalian metabolism are conserved in the fly. The major energy-storage organ of insects is the fat body (FB), a central metabolic tissue that exhibits physiological functions analogous to the mammalian adipose tissue and liver including nutrient storage, endocrine secretion, and immune response. Consequently, the FB makes intimate contact with both the gut where dietary nutrients re-absorbed and circulating hemolymph that transports lipids between organs. Drosophila larvae feed continuously to promote an increase in animal mass, and absorb dietary nutrients into the FB to store these as glycogen or triacylglyceride (TAG) that is incorporated into cytoplasmic lipid droplets (LDs). TAG storage ultimately requires LD biogenesis on the surface of the endoplasmic reticulum (ER), the primary site of TAG synthesis (Wilfling, 2014). During development or when nutrients are scarce, FB cells adapt their metabolism to mobilize LDs via cytoplasmic lipases. These mobilized lipids are delivered to other organs in the hemolymph via protein shuttles called lipophorin (Lpp) particles that are analogous to mammalian VLDL particles, but how LD mobilization is related to Lpp particle lipid loading remains poorly understood (Ugrankar, 2019).

The mechanisms that govern lipid flux across the FB cell plasma membrane (PM) also remain poorly characterized, but are essential for lipid export as well as lipid uptake and storage in LDs. In insects, the internalization of hemolymph lipids into both the FB and imaginal discs is unaffected when endocytosis is blocked, suggesting a non-vesicular uptake mechanism. In line with this, Lpp proteins are not degraded via endolysosomal trafficking within the FB, consistent with a model where Lpp particles can donate and receive lipids directly at the FB cell surface. Furthermore, Lpp particles primarily transport diacylglyceride (DAG), suggesting Lpp-derived lipids are processed during their uptake and delivery to the ER by ER-resident acyl CoA:diacylglycerol acyltransferase (DGAT) enzymes, which convert DAG to TAG. In addition to storing extracellular Lpp-derived lipids, FB cells also generate their own lipids via fatty acid de novo lipogenesis. FB cells deficient in fatty acid synthesis (FAS) enzymes exhibit severe lipodystrophy, indicating FB cells somehow balance the storage of Lpp-derived and de novo synthesized lipids to maintain fat homeostasis (Ugrankar, 2019).

Due to their specialized function in lipid uptake and storage, many fat-storing cells exhibit a unique surface architecture: their PM is densely pitted with invaginations that increase the surface area exposed to the extracellular space. In mammals, up to half the surface of white adipocytes is decorated with caveolae, invaginations that organize surface receptors as well as promote lipid and nutrient absorption. Surprisingly, Drosophila do not encode caveolin genes that are required to form caveolae. Nevertheless, Drosophila FB adipocytes exhibit their own intricate networks of surface invaginations that are stabilized by the cortical actin network. Perturbing this cortical actin network disrupts FB lipid homeostasis, suggesting a functional connection between FB surface architecture and lipid storage (Ugrankar, 2019).

Although LDs serve as organelle-scale lipid reservoirs, how cells organize their LD stores to balance storage with efficient mobilization is largely unresolved. An intuitive mechanism to organize LDs is to attach them to other organelles, as this allows them to exchange lipids with these organelles as well as potentially compartmentalize them in distinct regions of the cell interior. Recent work using Saccharomyces cerevisiae reveal that even simple yeast contain functionally distinct LD sub-populations that are spatially compartmentalized. This compartmentalization is achieved by LD-organizing proteins that bind to LDs and cluster them adjacent to the yeast vacuole/lysosome. One such organizing protein is Mdm1, an ER-anchored protein that binds to LDs and attaches them to the vacuole/lysosome surface. Mdm is highly conserved in Drosophila as CG1514/Snazarus (Snz), originally characterized as a longevity-associated gene of unknown function that is highly expressed in the Drosophila FB (Suh, 2008). Both yeast and human Snz homologs bind to LDs and regulate LD homeostasis, but the function of Snz in Drosophila remains unclear (Hariri, 2019; Datta, 2019). This study investigated how FB cells functionally and spatially organize their LD stores. FB cells contain functionally distinct LD populations that are spatially segregated into regions of the cell interior. These LD populations require distinct lipid pools for their maintenance, with LDs in the cell periphery (peripheral LDs, pLDs) requiring Lpp-dependent trafficking, whereas LDs further in the cell interior (medial LDs, mLDs) are maintained by FASN1-dependent de novo lipogenesis within the FB. Snz was also characterized as a novel regulator of pLD homeostasis that localizes to ER-PM contacts and promotes LD growth and TAG storage (Ugrankar, 2019).

Professional fat-storing cells must organize their fat reserves to balance long- term storage with the ability to efficiently mobilize lipids during energetic crises like starvation or metamorphosis. How this organization is achieved is unknown but presents significant spatial and metabolic challenges for the cell. This study reports that Drosophila FB adipocytes contain functionally distinct LD populations that are spatially segregated in the cell cytoplasm. A pLD population is maintained adjacent to the cell surface and makes intimate contact with the PM. pLD size and abundance are altered in response to fasting, suggesting pLDs are mobilized to provide circulating lipids for other larval tissues. Consistent with this, loss of lipophorin (Lpp) particles by ApoLppRNAi impacts pLD abundance and morphology, suggesting pLD maintenance requires some aspect of Lpp lipid trafficking. FB cells also contain a larger mLD population in the cell mid-plane region that is unaffected by loss of Lpp, but is drastically affected by loss of FASN1-mediated de novo lipogenesis in the FB. Remarkably, pLDs were still observed docked on the inner surface of the PM in FASN1RNAi FB tissue, further suggesting that pLDs contain lipids derived from extracellular sources that may be delivered into the FB via Lpp-dependent trafficking. This study also found that pLDs and mLDs are differentially dependent on perilipins, with mLDs relying on LSD for their morphology whereas pLDs require LSD2. Finally, this study has identified Snz as a LD-associated protein that is required for proper LD homeostasis in the FB. Snz localizes to ER-PM contacts in the FB cell periphery, and its over-expression increases TAG storage, consistent with a model whether Snz regulates ER-PM inter-organelle crosstalk that promotes lipid storage in LDs. In line with this, Snz functionally interacts with the ER- resident FA desaturase DESAT1, which is required for Snz-driven TAG accumulation (Ugrankar, 2019).

LDs have long been observed to be tethered to other organelles such as mitochondria and peroxisomes, and this impacts their sub-cellular distribution as well as their ability to exchange lipids with these organelles. Recent studies have identified specific proteins that bind to the surfaces of LDs, and mediate their attachment to other cellular organelles. Among these, yeast Mdm directly binds to nascent LDs, and promotes their attachment to the yeast vacuole. This Mdm1- vacuole interaction is critical for defining this LD positioning, as replacement of Mdm1's vacuole-binding PX domain with a PM-binding domain re-localizes Mdm to ER-PM contact sites and causes LDs to bud instead from the cortical ER adjacent to the PM (Hanaa Hariri, 2019). In addition to its role as an organelle tether, Mdm also positively regulates LD biogenesis by recruiting the fatty acyl-CoA ligase Faa to the ER surface, where it induces the incorporation of FAs into TAG in the LD (Hanaa Hariri, 2019). This study finds that Snz may function similarly to Mdm in both the spatial positioning of LDs, as well as promoting LD biogenesis. Snz localizes to the FB cell periphery and co-localizes with the ER-PM contact site biomarker dMAPPER, suggesting it enriches at regions of close contact between the ER and PM and potentially functions as an inter-organelle tether. Consistent with this, the Snz PX domain binds to liposomes containing phospholipids normally enriched on the PM, and exhibits a non-canonical lipid binding surface that mediates electrostatic interactions with phospholipids that would be present on the PM. This suggests a model where Snz functions in some aspect of functional coupling between the ER and PM, and potentially assists in lipid uptake from the hemolymph. Snz may also help to localize ER-resident lipid processing enzymes such as DESAT to the cell periphery, creating a localized pool of FA processing enzymes in the peripheral ER network which can efficiently process lipids prior to their incorporation into TAG. Consistent with this, Snz over-expression in the FB promotes TAG storage, and Snz can directly associate with LDs (Ugrankar, 2019).

Once thought to be pathogenic, adipocyte fat stores have more recently been proposed to act as metabolic buffers that protect against caloric overload and serve as sinks to reduce circulating lipids and sugars. As such, factors that enhance adipocyte fat storage may be protective against insulin insensitivity, organismal lipotoxicity, and other T2D-like pathologies. The data are consistent with this model, and indicate that Snz up-regulation promotes TAG storage in the FB through a DESAT1-dependent pathway. These elevated TAG stores not only prolong organismal survival during sustained fasting, but also promote organismal homeostasis that extends Drosophila lifespan, as well as buffers the pathological effects of chronic HSD (Ugrankar, 2019).

Collectively, these data support a model where Snz-associated pLDs provide a spatially-compartmentalized sink for lipids derived from the extracellular hemolymph. This pLD population may serve multiple functions. It could allow FB cells to quickly and efficiently process and store incoming Lpp-derived lipids from the hemolymph, thus avoiding their potentially lipotoxic accumulation in the cytoplasm. This would promote general cellular homeostasis and could minimize FA lipotoxicity during elevated lipid uptake. In addition, the high surface-to-volume size ratio of small pLDs may promote their efficient mobilization by cytoplasmic lipases during fasting. Since they are near the surface, pLD mobilization could also allow liberated FAs to be efficiently transferred to Lpp particles docked on the FB surface, where they can be subsequently trafficked to other organs. Snz has clear mammalian homologs including SNX which also bind PM-associated phospholipids. Whether SNX or other Snz homologs are also able to interact with LDs in distinct regions of the mammalian cell interior is unclear, but will be the focus of future studies (Ugrankar, 2019).

Snazarus and its human ortholog SNX25 modulate autophagic flux

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

An RGS-containing sorting nexin controls Drosophila lifespan

The pursuit of eternal youth has existed for centuries and recent data indicate that fat-storing tissues control lifespan. In a D. melanogaster fat body insertional mutagenic enhancer trap screen designed to isolate genes that control longevity, a regulator of G protein signaling (RGS) domain containing sorting nexin, termed snazaru (sorting nexin lazarus, snz), was identified. Flies with insertions into the 5' UTR of snz live up to twice as long as controls. Transgenic expression of UAS-Snz from the snz Gal4 enhancer trap insertion, active in fat metabolic tissues, rescued lifespan extension. Further, the lifespan extension of snz mutants was independent of endosymbiont, e.g., Wolbachia, effects. Notably, old snz mutant flies remain active and fertile indicating that snz mutants have prolonged youthfulness, a goal of aging research. Since mammals have snz-related genes, it is possible that the functions of the snz family may be conserved to humans (Suh, 2008).

For centuries humans have searched for keys to long-life and recent experiments in a variety of model systems support the notion that fat tissues are important in longevity. To identify regulators of lifespan expressed in sites that regulate metabolism, this study designed a multi-tiered approach, in which the initial step was a minimal promoter-Gal4; UAS-GFP fat body enhancer trap screen. The enhancer trap collection was examined for lifespan, selecting ten lines that had significantly increased longevity during multiple assays in both males and females. These data indicate that fat body mutant collections are a rich resource to identify genes important in lifespan control. A preliminary analyses indicate that the fat body enhancer trap screen did not approach saturation, so there are likely to be a diverse array of other genes that could be identified with related approaches directed towards metabolic tissues that could provide substantial insight into lifespan control. These could include additional F1 enhancer trap screens as well as other tiered longevity screens, for example first selecting for fat defects based upon buoyancy, starvation survival, or triglyceride content, might also be appropriate in the search for genes that regulate longevity (Suh, 2008).

Attention was focused on the C32 line because these flies had the greatest extension of lifespan of any line in the collection and because old C32 flies were active and fertile. C32 inserted into the 5'UTR of the hypothetical gene CG1514, predicted to encode an RGS domain containing sorting nexin, that was termed snazarus-sorting nexin lazarus (snz, pronounced snaz). Several lines of evidence support the notion that snz is responsible for the longevity phenotype. For example, inverse PCR from both 5' and 3' ends of C32 produce a single unique product, and only rone insertion was recovered in plasmid rescue experiments. Further, backcrossing the C32 insertion >10 generations into a w1118 background, to attempt to reduce both strain effects and second site mutations, maintained lifespan extension. To further examine the notion that snz was the responsible locus, the C32 P-element was mobilized and found that the longevity of the most precise excision lines reverted towards control. However, these excision strains had longer life than control w1118 flies, which may be secondary to background effects inherent in the methodology, the inability to backcross the revertants into w1118 due to the loss of the eye-color marker, the presence of the remaining piece of the P-element, local hopping of the P-element into other regions of the snz gene, or other factors. Notably, two other independently derived P-element insertions in the snz locus, G1409 and SZ4089, also conferred long life and display transheterozygous lifespan extension. These data indicate that the interval in which the P-elements are inserted confer the longevity phenotypes. However, there remains a possibility that snz is not the causative gene as the P-element insertions could alter linked genes or even have long-range effects, which could be clarified by identifying snz point mutations or with snz RNAi. An alternative approach was taken and determined whether Snz transgenesis could regulate lifespan. Transgenic expression of Snz reverses the C32 longevity phenotype, which indicates that Snz can alter lifespan. Thus the accumulated data are consistent with the idea that Snz regulates lifespan (Suh, 2008).

Snz is a member of the sorting nexin (Snx) family, defined by the presence of a PX, phospholipid binding domain. A general theme of the Snx family is that they regulate various aspects of endocytosis, important in internalization and in modulating signal transduction. Many mammalian Snxs direct trafficking of surface receptors including tyrosine kinase receptors, in some cases increasing and in others reducing signal transduction. Snz and the three related mammalian homologs, SNX13, SNX14, and SNX25, are a subgroup of the Snx family that, in addition to the signature PX domain, all contain an RGS domain indicating potential additional roles in signal transduction. RGS family proteins attenuate heterotrimeric G-protein signaling. The RGS domain of the Snz homolog SNX13 is unique among tested RGS domains in the ability to reduce signaling from Gαs proteins that regulate cAMP levels and thereby protein kinase A (PKA) action. A recent study showed that activating the PKA pathway increased lifespan. So, the RGS-containing Snx subgroup could control lifespan or metabolism by regulating protein trafficking and/or by modulating G protein signaling. Structure-function studies with Snz, such as attempting rescues with forms of Snz in which the PX or RGS domain is mutated, may help to clarify these notions (Suh, 2008).

Genetic studies have begun to identify mechanisms that regulate lifespan. These efforts have been hampered by the paucity of single gene mutants that display extended longevity. Recent data have raised the possibility that some of these few known mutants may not actually be long-lived. Rather, the longevity appears induced by a complex interaction with an intracellular bacterium and the phenotype can be completely reversed by treatment with the antibiotic tetracycline. Although this opens up the possibility to investigate genetic, environment, and flora interactions that may be important, they highlight the need to identify mutants that display longevity that is independent of bacterial contamination. Since the snz mutants have extended lifespan and enhanced fecundity, hallmarks of such infections, the three snz alleles were treated with the antibiotic tetracycline to evaluate possible dependence of the lifespan extension on the flora. However, even after the course of antibiotic therapy, all three snz mutants remained long-lived. Thus the mechanisms of lifespan extension conferred by reducing Snz action appear independent of tetracycline-sensitive microorganisms (Suh, 2008).

Studies of invertebrate mutants, and the responsible genes, have significantly contributed to mechanistic understanding of lifespan control. Remarkably, many of the pathways that control invertebrate lifespan also appear likely to have related functions in mammals. Many of these pathways are important in human health and especially in disorders of metabolism such as obesity and diabetes. For example, insulin signaling regulates invertebrate and vertebrate lifespan, and drugs that target this pathway are central diabetes therapies. Further, Sir2 controls yeast, worm, and fly longevity and small molecules that target Sir2 improved mammalian metabolic parameters such as blood glucose levels. This study has described a tiered, F1 strategy to identify flies with extended lifespan based upon enrichment for insertions in genes that are expressed in fat metabolic tissues. Given that relatively few single gene fly long-lived mutants have so far been identified, the data indicate that such collections are a rich resource to identify molecules important in lifespan control (Suh, 2008).

Functions of Snazarus orthologs in other species

Cerebellar ataxia disease-associated Snx14 promotes lipid droplet growth at ER-droplet contacts

Lipid droplets (LDs) are nutrient reservoirs used by cells to maintain homeostasis. Nascent droplets form on the endoplasmic reticulum (ER) and grow following an influx of exogenous fatty acids (FAs). The budding of LDs requires extensive ER-LD crosstalk, but how this is regulated remains poorly understood. This study shows that sorting nexin protein Snx14, an ER-resident protein associated with the cerebellar ataxia SCAR20, localizes to ER-LD contacts following FA treatment, where it promotes LD maturation. Using proximity-based APEX technology and topological dissection, this study shows that Snx14 accumulates specifically at ER-LD contacts independently of Seipin, where it remains ER-anchored and binds LDs in trans. SNX14(KO) cells exhibit perturbed LD morphology, whereas Snx14 overexpression promotes LD biogenesis and extends ER-LD contacts. Multi-time point imaging reveals that Snx14 is recruited to ER microdomains containing the fatty acyl-CoA ligase ACSL3, where nascent LDs bud. It is proposes that Snx14 is a novel marker for ER-LD contacts and regulates FA-stimulated LD growth (Datta, 2019).

Mdm1 maintains endoplasmic reticulum homeostasis by spatially regulating lipid droplet biogenesis

Lipid droplets (LDs) serve as cytoplasmic reservoirs for energy-rich fatty acids (FAs) stored in the form of triacylglycerides (TAGs). During nutrient stress, yeast LDs cluster adjacent to the vacuole/lysosome, but how this LD accumulation is coordinated remains poorly understood. The ER protein Mdm1 is a molecular tether that plays a role in clustering LDs during nutrient depletion, but its mechanism of function remains unknown. This study shows that Mdm1 associates with LDs through its hydrophobic N-terminal region, which is sufficient to demarcate sites for LD budding. Mdm1 binds FAs via its Phox-associated domain and coenriches with fatty acyl-coenzyme A ligase Faa1 at LD bud sites. Consistent with this, loss of MDM1 perturbs free FA activation and Dga1-dependent synthesis of TAGs, elevating the cellular FA level, which perturbs ER morphology and sensitizes yeast to FA-induced lipotoxicity. It is proposed that Mdm1 coordinates FA activation adjacent to the vacuole to promote LD production in response to stress, thus maintaining ER homeostasis (Hariri, 2019).

SNX14 mutations affect endoplasmic reticulum-associated neutral lipid metabolism in autosomal recessive spinocerebellar ataxia 20

Mutations in SNX14 cause the autosomal recessive cerebellar ataxia 20 (SCAR20). Mutations generally result in loss of protein although several coding region deletions have also been reported. Patient-derived fibroblasts show disrupted autophagy, but the precise function of SNX14 is unknown. The yeast homolog, Mdm1, functions in endoplasmic reticulum (ER)-lysosome/vacuole inter-organelle tethering, but functional conservation in mammals is still required. This study shows that loss of SNX14 alters but does not block autophagic flux. In addition, it was found that SNX14 is an ER-associated protein that functions in neutral lipid homeostasis and inter-organelle crosstalk. SNX14 requires its N-terminal transmembrane helices for ER localization, while the Phox homology (PX) domain is dispensable for subcellular localization. Both SNX14-mutant fibroblasts and SNX14KO HEK293 cells accumulate aberrant cytoplasmic vacuoles, suggesting defects in endolysosomal homeostasis. However, ER-late endosome/lysosome contact sites are maintained in SNX14KO cells, indicating that it is not a prerequisite for ER-endolysosomal tethering. Further investigation of SNX14- deficiency indicates general defects in neutral lipid metabolism. SNX14KO cells display distinct perinuclear accumulation of filipin in LAMP1-positive lysosomal structures indicating cholesterol accumulation. Consistent with this, SNX14KO cells display a slight but detectable decrease in cholesterol ester levels, which is exacerbated with U18666A. Finally, SNX14 associates with ER-derived lipid droplets (LD) following oleate treatment, indicating a role in ER-LD crosstalk. This study therefore has identified an important role for SNX14 in neutral lipid homeostasis between the ER, lysosomes and LDs that may provide an early intervention target to alleviate the clinical symptoms of SCAR20 (Bryant, 2018).

Mdm1/Snx13 is a novel ER-endolysosomal interorganelle tethering protein

Although endolysosomal trafficking is well defined, how it is regulated and coordinates with cellular metabolism is unclear. To identify genes governing endolysosomal dynamics, a global fluorescence-based screen was constructed to reveal endomembrane effector genes. Screening implicated Phox (PX) domain-containing protein Mdm1 in endomembrane dynamics. Surprisingly, it was demonstrated that Mdm1 is a novel interorganelle tethering protein that localizes to endoplasmic reticulum (ER)-vacuole/lysosome membrane contact sites (MCSs). Mdm1 is ER anchored and contacts the vacuole surface in trans via its lipid-binding PX domain. Strikingly, overexpression of Mdm1 induced ER-vacuole hypertethering, underscoring its role as an interorganelle tether. It was also shown that Mdm1 and its paralogue Ydr179w-a (named Nvj3 in this study) localize to ER-vacuole MCSs independently of established tether Nvj1. Finally, it was found that Mdm1 truncations analogous to neurological disease-associated SNX14 alleles fail to tether the ER and vacuole and perturb sphingolipid metabolism. This work suggests that human Mdm1 homologues may play previously unappreciated roles in interorganelle communication and lipid metabolism (Henne, 2015).

Structural basis for different phosphoinositide specificities of the PX domains of sorting nexins regulating G-protein signaling

Sorting nexins (SNXs) or phox homology (PX) domain containing proteins are central regulators of cell trafficking and signaling. A subfamily of PX domain proteins possesses two unique PX-associated domains, as well as a regulator of G protein-coupled receptor signaling (RGS) domain that attenuates Galphas-coupled G protein-coupled receptor signaling. This study has delineated the structural organization of these RGS-PX proteins, revealing a protein family with a modular architecture that is conserved in all eukaryotes. The one exception to this is mammalian SNX19, which lacks the typical RGS structure but preserves all other domains. The PX domain is a sensor of membrane phosphoinositide lipids, and this study has found that specific sequence alterations in the PX domains of the mammalian RGS-PX proteins, SNX13, SNX14, SNX19, and SNX25, confer differential phosphoinositide binding preferences. Although SNX13 and SNX19 PX domains bind the early endosomal lipid phosphatidylinositol 3-phosphate, SNX14 shows no membrane binding at all. Crystal structures of the SNX19 and SNX14 PX domains reveal key differences, with alterations in SNX14 leading to closure of the binding pocket to prevent phosphoinositide association. These findings suggest a role for alternative membrane interactions in spatial control of RGS-PX proteins in cell signaling and trafficking (Mas, 2014).


Search PubMed for articles about Drosophila Snazarus

Bryant, D., Liu, Y., Datta, S., Hariri, H., Seda, M., Anderson, G., Peskett, E., Demetriou, C., Sousa, S., Jenkins, D., Clayton, P., Bitner-Glindzicz, M., Moore, G. E., Henne, W. M. and Stanier, P. (2018). SNX14 mutations affect endoplasmic reticulum-associated neutral lipid metabolism in autosomal recessive spinocerebellar ataxia 20. Hum Mol Genet 27(11): 1927-1940. PubMed ID: 29635513

Datta, S., Liu, Y., Hariri, H., Bowerman, J. and Henne, W. M. (2019). Cerebellar ataxia disease-associated Snx14 promotes lipid droplet growth at ER-droplet contacts. J Cell Biol 218(4): 1335-1351. PubMed ID: 30765438

Hariri, H., Speer, N., Bowerman, J., Rogers, S., Fu, G., Reetz, E., Datta, S., Feathers, J. R., Ugrankar, R., Nicastro, D. and Henne, W. M. (2019). Mdm1 maintains endoplasmic reticulum homeostasis by spatially regulating lipid droplet biogenesis. J Cell Biol 218(4): 1319-1334. PubMed ID: 30808705

Henne, W. M., Zhu, L., Balogi, Z., Stefan, C., Pleiss, J. A. and Emr, S. D. (2015). Mdm1/Snx13 is a novel ER-endolysosomal interorganelle tethering protein. J Cell Biol 210(4): 541-551. PubMed ID: 26283797

Mas, C., Norwood, S. J., Bugarcic, A., Kinna, G., Leneva, N., Kovtun, O., Ghai, R., Ona Yanez, L. E., Davis, J. L., Teasdale, R. D. and Collins, B. M. (2014). Structural basis for different phosphoinositide specificities of the PX domains of sorting nexins regulating G-protein signaling. J Biol Chem 289(41): 28554-28568. PubMed ID: 25148684

Suh, J. M., Stenesen, D., Peters, J. M., Inoue, A., Cade, A. and Graff, J. M. (2008). An RGS-containing sorting nexin controls Drosophila lifespan. PLoS One 3(5): e2152. PubMed ID: 18478054

Ugrankar, R., Bowerman, J., Hariri, H., Chandra, M., Chen, K., Bossanyi, M. F., Datta, S., Rogers, S., Eckert, K. M., Vale, G., Victoria, A., Fresquez, J., McDonald, J. G., Jean, S., Collins, B. M. and Henne, W. M. (2019). Drosophila Snazarus regulates a lipid droplet population at plasma membrane-droplet contacts in adipocytes. Dev Cell 50(5): 557-572 e555. PubMed ID: 31422916

Wilfling, F., Thiam, A. R., Olarte, M. J., Wang, J., Beck, R., Gould, T. J., Allgeyer, E. S., Pincet, F., Bewersdorf, J., Farese, R. V., Jr. and Walther, T. C. (2014). Arf1/COPI machinery acts directly on lipid droplets and enables their connection to the ER for protein targeting. Elife 3: e01607. PubMed ID: 24497546

Biological Overview

date revised: 2 September 2022

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