InteractiveFly: GeneBrief

Autophagy-related 9: Biological Overview | References

BIOLOGICAL OVERVIEW

Autophagy is a highly conserved catabolic process that degrades and recycles intracellular components through the lysosomes. Atg9 is the only integral membrane protein among autophagy-related (Atg) proteins thought to carry the membrane source for forming autophagosomes. This study shows that Drosophila Atg9 interacts with Drosophila tumor necrosis factor receptor-associated factor 2 (dTRAF2: TNF-receptor-associated factor 6) to regulate the c-Jun N-terminal kinase (JNK) signaling pathway. Significantly, depletion of Atg9 and dTRAF2 compromised JNK-mediated intestinal stem cell proliferation and autophagy induction upon bacterial infection and oxidative stress stimulation. In mammalian cells, mAtg9 interacts with TRAF6, the homolog of dTRAF2, and plays an essential role in regulating oxidative stress-induced JNK activation. Moreover, it was found that ROS-induced autophagy acts as a negative feedback regulator of JNK activity by dissociating Atg9/mAtg9 from dTRAF2/TRAF6 in Drosophila and mammalian cells, respectively. These findings indicate a dual role for Atg9 in the regulation of JNK signaling and autophagy under oxidative stress conditions (Tang, 2013).

Macroautophagy (hereafter autophagy) is a conserved catabolic pathway in which double membrane vesicles called autophagosomes engulf macromolecules or organelles. Subsequently, autophagosomes fuse with lysosomes to form autolysosomes where degradation occurs. Autophagy is involved in cytoprotective responses to environmental stresses, stem cell maintenance and differentiation, tumorigenesis, and programmed cell death. There have been more than 30 autophagy-related (Atg) genes essential for autophagy process identified through genetic screens in yeast. Atg9 is the only one identified as a transmembrane protein, and it has been thought to promote lipid transport to the forming autophagosomes (Webber, 2010b). Mammalian Atg9 (mAtg9) localizes on the trans-Golgi network and endosomes under nutrient-rich conditions, whereas it translocates to forming autophagosomes under starvation conditions (Orsi, 2012; Young, 2006). The recycling of mAtg9 during autophagy is regulated by several proteins including Ulk1, ZIPK, mAtg13, and p38IP (Tang, 2011a; Webber; 2010a; Young, 2006). Interestingly, one recent study has reported that mAtg9 modulates innate immune response in an autophagy-independent manner (Saitoh, 2009). However, the physiological functions of Atg9 remain elusive (Tang, 2013).

Reactive oxygen species are highly reactive free radicals that can cause irreversible oxidative damage to proteins, lipids, or nucleotides in cells. Excessive production of ROS or depletion of antioxidants causes oxidative stress that often leads to cell dysfunction and diseases such as neurodegeneration, cancer, and aging. More importantly, ROS also plays critical roles in host defense and in the regulation of various cellular signaling pathways The ROS-induced signaling pathways include several mitogen-activated protein (MAP) kinase cascades involving the c-Jun NH2-terminal kinase (JNK) and p38 MAP kinase. The JNK signaling pathway regulates diverse biological functions, including apoptosis, cytoprotection, metabolism, and epithelial homeostasis in response to several cytokines and environmental stresses. Depending on the duration and magnitude of exposure, ROS-induced JNK activation may lead to the promotion of either cell survival or apoptosis. In Drosophila, JNK signaling was found to protect cells from oxidative stress and extend lifespan of adult flies. It has been shown that the JNK pathway is required for intestinal epithelium renewal during bacterial infection-induced ROS/oxidative stress. One of the mechanisms that JNK meditates to protect flies against acute oxidative insults is the activation of autophagy. In response to oxidative stress, JNK signaling stimulates the expression of several ATG genes (Wu, 2009). Several recent studies (Juhász, 2007; Simonsen, 2008) have reported that overexpression of ATG genes and activation of autophagy are sufficient to extend lifespan and confer stress resistance in Drosophila (Tang, 2013).

How does ROS/oxidative stress trigger JNK activation? It has been shown that signaling molecules, including apoptosis signal-regulating kinase (Ask1), glutathione S-transferase Pi (GSTp), and Src kinase can function as molecular links between ROS and JNK (Shen, 2006). Ask1 is a MAPKKK that activates JNK by phosphorylating MKK4/7. Under normal physiological conditions, Ask1 is inhibited by forming a complex with the redox regulatory protein thioredoxin. Upon exposure to ROS/oxidative stress, the oxidized thioredoxin dissociates from Ask1 and results in the activation of Ask1 signaling pathway. GSTp has been identified as a JNK inhibitor. Under oxidative conditions, GSTp forms oligomers and dissociates from JNK, leading to JNK activation. A number of reports have also shown the involvement of Src and its downstream targets in H2O2-induced JNK activation, although the underlying molecular mechanism remains elusive. Recently, tumor necrosis factor receptor-associated factors (TRAFs) have been found to be involved in ROS-mediated JNK activation. In mammals, the TRAF family consists of seven members and functions as scaffold proteins that link cell surface receptors to the downstream effectors. Among them, TRAF2 and TRAF6 are found to associate with Ask1 and form the active Ask1 signalsome in response to ROS stimulation (Fujino, 2007 and Noguchi, 2005). Moreover, the involvement of TRAF4 in oxidative activation of JNK via its interaction with the NAD(P)H oxidase p47phox has been demonstrated (Xu, 2002). The Drosophila TRAF2 (dTRAF2), a homolog of human TRAF6, was found to mediate Eiger/Wegen (tumor necrosis factor/tumor necrosis factor receptor [TNF/TNFR])-induced JNK signaling (Xue, 2007). However, the role of dTRAF2 in ROS-mediated JNK activation remains unclear (Tang, 2013).

This study has identified a biological function of Atg9 in regulation of JNK signaling pathway. Drosophila Atg9 can activate JNK signaling through its interaction with dTRAF2. Depletion of Atg9 compromised oxidative stress-induced JNK activation, the JNK-mediated epithelium renewal, and autophagy induction. In mammalian cells, mAtg9 was found to be essential for JNK activation in response to ROS/oxidative stress, indicating a highly conserved role of Atg9 in regulating JNK activity. It was further found that ROS-induced autophagy negative feedback regulates JNK activity through the dissociation of Atg9/mAtg9 from dTRAF2/TRAF6 in Drosophila and mammalian cells, respectively. These findings provide insights into the crosstalk between autophagy and JNK signaling pathway in response to oxidative stress (Tang, 2013).

The Atg9 transmembrane protein has been shown to play an essential role in autophagy pathway in yeast and mammals (Webber, 2010b). In this study, Drosophila Atg9 was also found to be required for autophagy induction upon nutrient deprivation or under oxidative stress conditions. More importantly, a role was uncovered for Atg9 in regulating the JNK signaling pathway. Upon bacterial infection, Atg9 interacts with dTRAF2 to activate JNK-mediated autophagy induction and epithelium renewal in Drosophila gut cells. The role of Atg9 in activating JNK signaling was also observed in mammalian cells. Moreover, this study found that ROS-induced autophagy in turn inhibits JNK signaling via a negative feedback mechanism by dissociation of Atg9 from dTRAF2 and TRAF6 in Drosophila and mammalian cells, respectively (Tang, 2013).

Atg9 is a highly conserved and the only multi-spanning transmembrane Atg protein essential for the formation of autophagosomes. In yeast, Atg9 cycles between the preautophagosomal structure (PAS) and peripheral cytoplasmic structures (Mari, 2007). Recently, using single particle tracking, Yamamoto (2012) found that yeast Atg9 exists as highly motile vesicles that contribute to PAS formation. In mammalian cells, mAtg9 is localized mainly to the trans-Golgi network and endosomes (Young, 2006). However, upon nutrient starvation, mAtg9 is enriched in endosomal pools and undergoes a dynamic interaction with forming autophagosomes (Orsi, 2012). The current study found that Drosophila Atg9 not only distributed in cytoplasm, but also concentrated at cell-cell junctions, suggesting Atg9 may have additional roles besides its function in autophagy. For example, it has been reported that mAtg9 can function as a regulator for dsDNA-triggered innate immune response (Tang, 2013).

The involvement of Atg1/Ulk1 in Atg9 trafficking has been described in yeast and mammalian cells. Consistent with these findings, the current study found that Drosophila Atg9 redistributed from peripheral pools to forming autophagosomes in an Atg1-dependent manner. A previous reported that overexpression of Drosophila Atg1 induces cell death (Scott, 2007). Interestingly, this study found that overexpression of Atg1 did not induce JNK activation and the Atg1-induced cell death could not be rescued by inhibition of JNK signaling. The current findings highlight that, in addition to its role in autophagy, Atg9 plays a role in the regulation of JNK activation in response to oxidative stress (Tang, 2013).

The JNK signaling pathway is one of the mitogen-activated protein kinase (MAPK) cascades involved in stress responses. Activation of the JNK pathway has been implicated in a number of biological processes including cell proliferation, survival, apoptosis, and migration. The involvement of JNK in both proapoptotic and anti-apoptotic activities indicates a complex function of the JNK pathway, whereas the molecular mechanism that regulates JNK to mediate both processes remains elusive. This study study has shown that ectopic expression of Atg9 in the developing wing and eye leads to JNK activation and apoptotic cell death. Moreover, the results provided evidence that, upon ROS stimulation, Atg9, but not Atg12, is required for JNK-mediated intestinal stem cell proliferation and autophagy induction in Drosophila. These results indicate that Atg9 may play a critical role in regulating JNK-mediated cell survival and apoptosis. It was further shown that Atg9 regulates JNK signaling via its association with dTRAF2 and TRAF6 in Drosophila and mammals, respectively. GST-pull down assay revealed that the C terminus of Drosophila Atg9 can interact with dTRAF2. Surprisingly, Atg9 lacking the C-terminal region can still promote JNK activation and cell death. One possibility is that Atg9 may interact with dTRAF2 through multi-regions. On the other hand, yeast Atg9 has been shown to self-interact through the C terminus, and Atg9 self-association is critical for its function in autophagy (He, 2008). Sequence analysis revealed that Drosophila Atg9 also contains the conserved self-interacting motif (VGNVC) between amino acids 560 and 564. It is possible that Atg9ΔC may exert its function in regulating JNK activity by interacting with the endogenous Atg9 (Tang, 2013).

TRAF6 functions as a RING-domain containing ubiquitin ligase involved in a variety of biological processes including adaptive and innate immunity, bone metabolism and tissue development (Inoue, 2007 and Pineda, 2007). TRAF6 is required for interleukin-1 (IL-1) and transforming growth factor-β-mediated JNK activation (O'Neill, 2002, Sorrentino, 2008; Yamashita, 2008). In Drosophila, dTRAF2 plays a role in Eiger/Wegen (TNF/TNFR)-induced JNK signaling (Xue, 2007). How does Atg9 regulate TRAF-mediated JNK activation? One mechanism may be that Atg9 associate with TRAF6 to modulate its ubiquitin ligase activity. Indeed, a recent study indicates that Atg9 interacts and promotes TRAF6 ubiquitination. Alternatively, because Atg9 is a membrane protein with diverse subcellular localization, Atg9 may bind and target TRAF6 to peripheral membrane regions in response to bacterial infection and oxidative stress. These two mechanisms need not be mutually exclusive and can occur together (Tang, 2013).

Recent studies suggested there to be a complex relationship between the JNK pathway and autophagy. On the one hand, under nutrient starvation conditions, JNK has been found to phosphorylate Bcl-2, leading to the dissociation of Bcl-2 from beclin 1 and the activation of autophagy. JNK signaling also activates autophagy via the upregulation of ATG gene expression in response to oxidative stress and oncogenic transformation. On the other hand, JNK can act as a negative regulator of FoxO-dependent autophagy in neurons. It is interesting to note that, although Atg9 overexpression activates JNK, the current data showed that Atg9 overexpression could not induce autophagy in the larval fat body. Because Atg9 promotes JNK activation through its association with dTRAF2, dTRAF2 may not be expressed in the fat body. Indeed, RNA expression analysis reveals that dTRAF2 expresses in the fat body at a relatively low level . Alternatively, it has been reported that JNK overexpression activates autophagy independently of Atg1 and nutrient signal. However, the current results showed that Atg9 interacts with Atg1 and is required for starvation-induced autophagy. Overexpression of JNK may induce a noncanonical autophagy that is independent of 'core Atg proteins.' (Tang, 2013 and references therein).

This current study also demonstrates that autophagy can act as a negative feedback regulator for JNK activation upon oxidative stress. Inhibition of autophagy in flies fed with Ecc15 or paraquat resulted in a substantial increase in JNK activity, which led to increased ISC proliferation and cell death in adult Drosophila midgut. In mammalian cells, depletion of Atg5 led to prolonged JNK activation during hydrogen peroxide-induced oxidative stress. Moreover, activation of autophagy by rapamycin effectively blocked the interaction between Atg9 and TRAF6 and inhibits ROS-induced JNK activity. Considered together, these findings together indicate an important role of autophagy in restricting JNK activity by modulating the interaction between Atg9 and TRAF6 in response to oxidative stress. In conclusion, this work establishes a regulatory mechanism between Atg9, autophagy, and the JNK signaling pathway during oxidative stress conditions (Tang, 2013).

ESCRT dysfunction compromises endoplasmic reticulum maturation and autophagosome biogenesis in Drosophila

Autophagy targets cytoplasmic materials for degradation and influences cell health. Organelle contact and trafficking systems provide membranes for autophagosome formation, but how different membrane systems are selected for use during autophagy remains unclear. This study reports a novel function of the endosomal sorting complex required for transport (ESCRT) in the regulation of endoplasmic reticulum (ER) coat protein complex II (COPII) vesicle formation that influences autophagy. The ESCRT functions in a pathway upstream of Vps13D to influence COPII vesicle transport, ER-Golgi intermediate compartment (ERGIC) assembly, and autophagosome formation. Atg9 functions downstream of the ESCRT to facilitate ERGIC and autophagosome formation. Interestingly, cells lacking either ESCRT or Vps13D functions exhibit dilated ER structures that are similar to cranio-lenticulo-sutural dysplasia patient cells with SEC23A mutations, which encodes a component of COPII vesicles. These data reveal a novel ESCRT-dependent pathway that influences the ERGIC and autophagosome formation (Wang, 2022).

The ESCRT regulates autophagy in multiple ways, including AP membrane sealing and autolysosome (ALY) formation. This study reveals a previously undescribed function of the ESCRT in ER trafficking and autophagy. The findings indicate that the ESCRT is required for COPII vesicle-derived compartments and AP formation. Loss of ESCRT components influence COPII vesicle marker formation and ER morphology. This study also shows that Vps13D co-localizes with markers of different stages of COPII vesicle maturation between the ER and Golgi apparatus, including markers of ERGIC formation. In the absence of ESCRT and Vps13D function, ER structure is altered in a manner that is similar to CLSD patient cells with SEC23A mutations. In addition, the ESCRT impacts Atg9 distribution. Vps13D and Atg9 are required for autophagy and appear to influence the ERGIC for the induction of autophagy. Therefore, this study identifies previously unrecognized steps in a regulatory pathway for autophagy, including novel roles for the ESCRT and Vps13D in COPII-derived and ERGIC-associated AP formation (Wang, 2022).

Autophagy involves a core mechanism, but some autophagy regulatory mechanisms vary in different cell contexts in animals. During Drosophila midgut development, most of the core autophagy genes are required, but Atg7 and Atg3 are not required for cell size reduction, protein, and organelle clearance. Instead, the ubiquitin activating enzyme Uba1 and ubiquitin function in autophagy. Vps13D contains a conserved ubiquitin-binding UBA domain and is required for developmental autophagy in the fly intestine. Thus, Vps13D has properties of an autophagy receptor and therefore may link ubiquitin, autophagy proteins, and their autophagic cargo substrates. Alternatively, ubiquitin may have cargo-receptor-independent functions in autophagy during intestine development (Wang, 2022).

Tsg101 and Vps36 both possess ubiquitin-binding domains and are required for autophagy in the fly intestine. While previous studies have shown that the ESCRT functions in AP membrane sealing and ALY formation, studies of Tsg101, Vps36, and all other ESCRT components tested indicate a novel role in ER trafficking for AP formation in the intestine. This study systematically compared ESCRT function in different cell contexts in Drosophila. Consistent with previous studies, ESCRT mutant fat body cells accumulate Atg8a puncta indicating that autophagy is blocked following ALY formation. By contrast, the ESCRT is required for Atg8a puncta formation in intestine cells. Therefore, the ESCRT machinery functions in a cell context-specific manner to regulate autophagy (Wang, 2022).

ESCRT machinery regulates multiple dynamic membrane changes, including vesicle abscission, membrane sealing, and repair. The function of the ESCRT in the COPII pathway is novel but also has similarities to the essential function of the ESCRT in membrane remodeling. During Drosophila development, Hrs is distributed near ER and co-localized with Ergic53 when autophagy is induced. In ESCRT mutant cells, both COPII and Ergic53 puncta were decreased and dissociated, indicating that COPII vesicles derived from ER are inhibited without the ESCRT. Multiple membrane remodeling processes require the ESCRT, including vesicle budding and internalization. Thus, it appears that the ESCRT affects COPII-coated ER exit sites to shed vesicles and influences ERGIC and AP assembly in a cell-specific manner. Decreased function of ESCRT regulatory genes influences Vps13D levels. By contrast, Vps13D regulates COPII regulatory protein localization, suggesting that Vps13D functions downstream of the ESCRT to influence COPII. It should be noted, however, that it is difficult to exclude the possibility that the ESCRT may also participate in either AP membrane sealing or fusion between AP and lysosome at a later stage in the autophagy process. In addition, we cannot exclude a possible indirect influence of the ESCRT on the ER and COPII maturation that is independent of Vps34 (Wang, 2022).

The roles of the regulators of COPII vesicle and ERGIC formation in autophagy may be Drosophila intestine cell context specific. However, COPII and the ERGIC have been implicated in the regulation of autophagy in both yeast and mammalian cells. Atg9 interacts with membrane compartments from the secretory pathway, endocytic vesicles, and Golgi apparatus in both yeast and mammals. Atg9 interacts with the COPII component Sec24 in yeast, and co-fractionates with ERGIC components in mammalian cells. Thus, it is likely that membranes from COPII compartments are utilized for autophagy induction. Atg9 is conserved in Drosophila, and the current analyses indicate that Atg9 is downstream of the ESCRT, Vps13D, and the ERGIC. Therefore, the ERGIC is also a potential membrane source for Atg9 to regulate the induction of autophagy in Drosophila, consistent with ERGIC function in mammalian cells. Thus, it is possible that the roles of COPII, Vps13D, and the ERGIC are more prevalent in the regulation of autophagy than previously recognized. Future studies should reveal how broadly these factors function in autophagy, as well as the role of the ESCRT in COPII vesicle maturation in other cell types (Wang, 2022).

Atg1-mediated myosin II activation regulates autophagosome formation during starvation-induced autophagy

Autophagy is a membrane-mediated degradation process of macromolecule recycling. Although the formation of double-membrane degradation vesicles (autophagosomes) is known to have a central role in autophagy, the mechanism underlying this process remains elusive. The serine/threonine kinase Atg1 has a key role in the induction of autophagy. This study shows that overexpression of Drosophila Atg1 promotes the phosphorylation-dependent activation of the actin-associated motor protein myosin II. A novel myosin light chain kinase (MLCK)-like protein, Spaghetti-squash activator (Sqa), was identified as a link between Atg1 and actomyosin activation. Sqa interacts with Atg1 through its kinase domain and is a substrate of Atg1. Significantly, myosin II inhibition or depletion of Sqa compromised the formation of autophagosomes under starvation conditions. In mammalian cells, it was found that the Sqa mammalian homologue zipper-interacting protein kinase (ZIPK) and myosin II had a critical role in the regulation of starvation-induced autophagy and mammalian Atg9 (mAtg9) trafficking when cells were deprived of nutrients. These findings provide evidence of a link between Atg1 and the control of Atg9-mediated autophagosome formation through the myosin II motor protein (Tang, 2011a).

Myosin II is a conventional two-headed myosin composed of two heavy chains, two essential light chains, and two regulatory light chains. Myosin II activation is regulated by the phosphorylation of its regulatory light chain via MLCKs. Rho GTPase and Rho kinase have been implicated in the regulation of myosin activation. However, this study found that neither RNA-mediated knockdown of dRok nor mutations in Rho1 or dRhoGEF2 could suppress the Atg1-induced wing defects. Instead, it was found that depletion of Sqa rescued Atg1-induced wing defects. This epistasis analysis showed that Sqa functioned downstream of Atg1. Moreover, it was found that Sqa but not Atg1 could directly phosphorylate Spaghetti squash (Sqh) in the in vitro kinase assay, suggesting that Atg1 stimulates myosin activity via Sqa. Importantly, Atg1 phosphorylates and interacts with Sqa, indicating that Atg1-Sqa functions in a kinase cascade to regulate myosin II activation. Moreover, Atg1 has been found to have a critical role in the regulation of autophagy induction under stress conditions in yeast, Drosophila, and mammalian cells. These results provide the first evidence that nutrient starvation stimulates myosin II activation in an Atg1-Sqa-dependent manner. Most significantly, a dramatic decrease was found in the size and number of autophagosomes in cells expressing Sqa-T279A, Sqa-RNAi, and SqhA20A21 on nutrient deprivation, indicating that Atg1-Sqa-mediated actomyosin activation has a critical role in autophagy (Tang, 2011a).

The kinase domain of Sqa is also highly homologous to that of the mammalian DAPK family proteins. Recent studies have indicated that DAPK1 regulates autophagy through its association with MAP1B and Beclin1, or by modulating the Tor signalling pathway. As DAPK family proteins also regulate myosin II phosphorylation, one might speculate that Sqa may be the Drosophila counterpart of DAPK protein. Indeed, although overexpression of Sqa does not induce cell death, Sqa shares several characteristics with DAPK3/ZIPK. First, unlike MLCK family proteins, both Sqa and ZIPK contain an amino-terminal kinase domain that has 42% sequence identity and 61% similarity. Moreover, like ZIPK, recent sequence analysis from FlyBase identified a Sqa isoform that also contains a leucine-zipper domain. Second, as phosphorylation of Thr-265 in ZIPK is essential for its kinase activity, this study found that Atg1 phosphorylates Sqa at the corresponding Thr-279, and is critical for Sqa activity. Third, just as Sqa specifically associates with kinase-inactive Atg1, the results indicate a similar interaction between ZIPK and Ulk1. Importantly, depletion of Sqa and ZIPK resulted in autophagic defects in response to nutrient deprivation. These findings together suggest that ZIPK may act as a mammalian homolog of Sqa during starvation-induced autophagy. Further investigation is needed to determine whether the mammalian Atg1 (Ulk1) directly phosphorylates ZIPK at Thr-265, and the role of this regulation in autophagy (Tang, 2011a).

In autophagy, the source of the autophagosomal membrane and dynamics of autophagosome formation are fundamental questions. Studies in yeast and mammalian cells have identified several intracellular compartments as potential sources for the PAS (also termed isolation membrane/phagophore). Formation of PI(3)P-enriched ER subdomains (omegasomes) has been reported during nutrient starvation and autophagy induction, and a direct connection has been observed between ER and the phagophore using the 3D electron tomography. In addition, recent studies in yeast cells have suggested Atg9 and the Golgi complex have a role in the formation of autophagosomes. It has been proposed that the integral membrane protein Atg9 may respond to the induction signal in promoting lipid transport to the forming autophagosomes. The mAtg9 has been found to localize on the TGN and the endosomes in nutrient-rich conditions and translocate to LC3-positive autophagosomes on nutrient deprivation. Although several proteins, including Ulk1, mAtg13, and p38IP, have been found to regulate starvation-induced mAtg9 trafficking, the molecular motor that controls the movement of mAtg9 between different subcellular compartments remains unknown (Tang, 2011a).

The finding that myosin II redistributes from peripheral to the perinuclear region of cells on starvation suggests that myosin II has a role in membrane trafficking. In fact, it has been reported that myosin II is required for the trafficking of major histocompatibility complex (MHC) class II molecules and antigen presentation in B lymphocytes. Myosin II has also been found to be involved in the protein transport between ER and Golgi. This study has shown that there here is a molecule link between mAtg9 and the actomyosin network, indicating that myosin II may function as a motor protein for mAtg9 trafficking during early autophagosome formation. In conclusion, this work has unravelled a regulatory mechanism between Atg1 activity and the Atg9-mediated formation of autophagosomes. Further studies are needed to determine the involvement of this signalling process in other stress-induced or developmentally regulated autophagy (Tang, 2011a).

Unraveling the role of myosin in forming autophagosomes

Macroautophagy (hereafter autophagy) is a membrane-mediated catabolic process that occurs in response to a variety of intra- and extra-cellular stresses. It is characterized by the formation of specialized double-membrane vesicles, autophagosomes, which engulf organelles and long-lived proteins, and in turn fuse with lysosomes for degradation and recycling. How autophagosomes emerge is still unclear. The Atg1 kinase plays a crucial role in the induction of autophagosome formation. While several Atg (autophagy-related) proteins have been associated with, and have been found to regulate, Atg1 kinase activity, the downstream targets of Atg1 that trigger autophagy remain unknown. Recent studies have identified a myosin light chain kinase (MLCK)-like kinase as the Atg1 kinase effector that induces the activation of myosin II and have found it to be required for autophagosome formation during nutrient deprivation. It was further demonstrated that Atg1-mediated myosin II activation is crucial for the movement of the Atg9 transmembrane protein between the Golgi and the forming autophagosome, which provides a membrane source for the formation of autophagosomes during starvation (Tang, 2011b).

Impaired proteasomal degradation enhances autophagy via hypoxia signaling in Drosophila

Two pathways are responsible for the majority of regulated protein catabolism in eukaryotic cells: the ubiquitin-proteasome system (UPS) and lysosomal self-degradation through autophagy. Both processes are necessary for cellular homeostasis by ensuring continuous turnover and quality control of most intracellular proteins. Recent studies established that both UPS and autophagy are capable of selectively eliminating ubiquitinated proteins and that autophagy may partially compensate for the lack of proteasomal degradation, but the molecular links between these pathways are poorly characterized. This study shows that autophagy is enhanced by the silencing of genes encoding various proteasome subunits (alpha, beta or regulatory) in larval fat body cells. Proteasome inactivation induces canonical autophagy, as it depends on core autophagy genes Atg1, Vps34, Atg9, Atg4 and Atg12. Large-scale accumulation of aggregates containing p62 and ubiquitinated proteins is observed in proteasome RNAi cells. Importantly, overexpressed Atg8a reporters are captured into the cytoplasmic aggregates, but these do not represent autophagosomes. Loss of p62 does not block autophagy upregulation upon proteasome impairment, suggesting that compensatory autophagy is not simply due to the buildup of excess cargo. One of the best characterized substrates of UPS is the α subunit of hypoxia-inducible transcription factor 1 (HIF-1α), which is continuously degraded by the proteasome during normoxic conditions. Hypoxia is a known trigger of autophagy in mammalian cells, and this study shows that genetic activation of hypoxia signaling also induces autophagy in Drosophila. Moreover, it was found that proteasome inactivation-induced autophagy requires sima, the Drosophila ortholog of HIF-1alpha. This study has characterized proteasome inactivation- and hypoxia signaling-induced autophagy in the commonly used larval Drosophila fat body model. Activation of both autophagy and hypoxia signaling was implicated in various cancers, and mutations affecting genes encoding UPS enzymes have recently been suggested to cause renal cancer. These studies identify a novel genetic link that may play an important role in that context, as HIF-1alpha/sima may contribute to upregulation of autophagy by impaired proteasomal activity (Low, 2013).

Functions of Atg9 orthologs in other species

KIF1A/UNC-104 transports ATG-9 to regulate neurodevelopment and autophagy at synapses

Autophagy is a cellular degradation process important for neuronal development and survival. Neurons are highly polarized cells in which autophagosome biogenesis is spatially compartmentalized. The mechanisms and physiological importance of this spatial compartmentalization of autophagy in the neuronal development of living animals are not well understood. This study determines that, in Caenorhabditis elegans neurons, autophagosomes form near synapses and are required for neurodevelopment. It was shown through unbiased genetic screens and systematic genetic analyses that autophagy is required cell autonomously for presynaptic assembly and for axon outgrowth dynamics in specific neurons. Autophagosome biogenesis in the axon near synapses was observed, and this localization was found to depend on the synaptic vesicle kinesin, KIF1A/UNC-104 (see Drosophila Unc-104). KIF1A/UNC-104 coordinates localized autophagosome formation by regulating the transport of the integral membrane autophagy protein ATG-9 (see Drosophila Atg9). These findings indicate that autophagy is spatially regulated in neurons through the transport of ATG-9 by KIF1A/UNC-104 to regulate neurodevelopment (Stavoe, 2016).

Search PubMed for articles about Drosophila Atg9

Fujino, G., Noguchi, T., Matsuzawa, A., Yamauchi, S., Saitoh, M., Takeda, K. and Ichijo, H. (2007). Thioredoxin and TRAF family proteins regulate reactive oxygen species-dependent activation of ASK1 through reciprocal modulation of the N-terminal homophilic interaction of ASK1. Mol Cell Biol 27: 8152-8163. PubMed ID: 17724081

He, C., Baba, M., Cao, Y. and Klionsky, D. J. (2008). Self-interaction is critical for Atg9 transport and function at the phagophore assembly site during autophagy. Mol Biol Cell 19: 5506-5516. PubMed ID: 18829864

Inoue, J., Gohda, J., and Akiyama, T. (2007). Characteristics and biological functions of TRAF6. Adv. Exp. Med. Biol. 597: 72-79. PubMed ID: 17633018

Juhasz, G., Erdi, B., Sass, M. and Neufeld, T. P. (2007). Atg7-dependent autophagy promotes neuronal health, stress tolerance, and longevity but is dispensable for metamorphosis in Drosophila. Genes Dev 21: 3061-3066. PubMed ID: 18056421

Low, P., Varga, A., Pircs, K., Nagy, P., Szatmari, Z., Sass, M. and Juhasz, G. (2013). Impaired proteasomal degradation enhances autophagy via hypoxia signaling in Drosophila. BMC Cell Biol 14: 29. PubMed ID: 23800266

Mari, M. and Reggiori, F. (2007). Atg9 trafficking in the yeast Saccharomyces cerevisiae. Autophagy 3: 145-148. PubMed ID: 17204846

Noguchi, T., Takeda, K., Matsuzawa, A., Saegusa, K., Nakano, H., Gohda, J., Inoue, J. and Ichijo, H. (2005). Recruitment of tumor necrosis factor receptor-associated factor family proteins to apoptosis signal-regulating kinase 1 signalosome is essential for oxidative stress-induced cell death. J Biol Chem 280: 37033-37040. PubMed ID: 16129676

O'Neill, L. A. (2002). Signal transduction pathways activated by the IL-1 receptor/toll-like receptor superfamily. Curr Top Microbiol Immunol 270: 47-61. PubMed ID: 12467243

Orsi, A., Razi, M., Dooley, H. C., Robinson, D., Weston, A. E., Collinson, L. M. and Tooze, S. A. (2012). Dynamic and transient interactions of Atg9 with autophagosomes, but not membrane integration, are required for autophagy. Mol Biol Cell 23: 1860-1873. PubMed ID: 22456507

Pineda, G., Ea, C. K. and Chen, Z. J. (2007). Ubiquitination and TRAF signaling. Adv Exp Med Biol 597: 80-92. PubMed ID: 17633019

Saitoh, T., Fujita, N., Hayashi, T., Takahara, K., Satoh, T., Lee, H., Matsunaga, K., Kageyama, S., Omori, H., Noda, T., Yamamoto, N., Kawai, T., Ishii, K., Takeuchi, O., Yoshimori, T. and Akira, S. (2009). Atg9a controls dsDNA-driven dynamic translocation of STING and the innate immune response. Proc Natl Acad Sci U S A 106: 20842-20846. PubMed ID: 19926846

Shen, H. M. and Liu, Z. G. (2006). JNK signaling pathway is a key modulator in cell death mediated by reactive oxygen and nitrogen species. Free Radic Biol Med 40: 928-939. PubMed ID: 16540388

Simonsen, A., Cumming, R. C., Brech, A., Isakson, P., Schubert, D. R. and Finley, K. D. (2008). Promoting basal levels of autophagy in the nervous system enhances longevity and oxidant resistance in adult Drosophila. Autophagy 4: 176-184. PubMed ID: 18059160

Scott, R. C., Juhasz, G. and Neufeld, T. P. (2007). Direct induction of autophagy by Atg1 inhibits cell growth and induces apoptotic cell death. Curr Biol 17: 1-11. PubMed ID: 17208179

Sorrentino, A., Thakur, N., Grimsby, S., Marcusson, A., von Bulow, V., Schuster, N., Zhang, S., Heldin, C. H. and Landstrom, M. (2008). The type I TGF-beta receptor engages TRAF6 to activate TAK1 in a receptor kinase-independent manner. Nat Cell Biol 10: 1199-1207. PubMed ID: 18758450

Stavoe, A.K., Hill, S.E., Hall, D.H. and Colón-Ramos, D.A. (2016). KIF1A/UNC-104 transports ATG-9 to regulate neurodevelopment and autophagy at synapses. Dev Cell 38: 171-185. PubMed ID: 27396362

Tang, H. W., Wang, Y. B., Wang, S. L., Wu, M. H., Lin, S. Y. and Chen, G. C. (2011a). Atg1-mediated myosin II activation regulates autophagosome formation during starvation-induced autophagy. EMBO J 30: 636-651. PubMed ID: 21169990

Tang, H. W. and Chen, G. C. (2011b). Unraveling the role of myosin in forming autophagosomes. Autophagy 7: 778-779. PubMed ID: 21460626

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date revised: 5 August 2023

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