UV-resistance associated gene: Biological Overview | References
Gene name -
UV-resistance associated gene
Synonyms - Vps38
Cytological map position - 34B4-34B4
Function - signaling
Keywords - PI3K-cIII complex, autophagy, downregulation of Patched via the endosomal-lysosomal pathway to execute axon pruning, tumor suppressor, receptor downregulation through endolysosomal degradation, the establishment of proper cell polarity in the developing wing, UV-induced DNA damage repair
Symbol - Uvrag
FlyBase ID: FBgn0032499
Genetic map position - chr2L:13,286,777-13,289,556
Classification - Vacuolar sorting 38 and autophagy-related subunit 14
Cellular location - cytoplasmic
Developmental axon pruning is essential for wiring the mature nervous system, but its regulation remains poorly understood. This study shows that the endosomal-lysosomal pathway regulates developmental pruning of Drosophila mushroom body γ neurons. The UV radiation resistance-associated gene (Uvrag) functions together with all core components of the phosphatidylinositol 3-kinase class III (PI3K-cIII) complex to promote pruning via the endocytic pathway. By studying several PI3P binding proteins, this study found that Hrs, a subunit of the ESCRT-0 complex, required for multivesicular body (MVB) maturation, is essential for normal pruning progression. Thus, the existence of an inhibitory signal that needs to be downregulated is hypothesized. Finally, the data suggest that the Hedgehog receptor, Patched, is the source of this inhibitory signal likely functioning in a Smo-independent manner. Taken together, this in vivo study demonstrates that the PI3K-cIII complex is essential for downregulating Patched via the endosomal-lysosomal pathway to execute axon pruning (Issman-Zecharya, 2014).
Neuronal remodeling is an essential step of nervous system development in both vertebrates and invertebrates. One mechanism used to remodel neuronal circuits is by the elimination of long stretches of axons in a process known as axon pruning. With a few exceptions, the current dogma is that axon pruning of long stretches of axons occurs via local axon degeneration while axon pruning of short stretches occurs via retraction. While in some cases remodeling is directly affected by experience or neural activity, in cases of stereotypical pruning the identity of the axon that is destined to be pruned does not depend on experience or neural activity. Because of mechanistic similarities to Wallerian degeneration and dying back neurodegenerative diseases, understanding the molecular mechanisms of axon pruning should result in a broader insight into axon fragmentation and elimination during development and in disease (Issman-Zecharya, 2014).
The neuronal remodeling of the Drosophila mushroom body (MB) during development is a unique model system to study the molecular aspects of axon pruning. The stereotypic temporal and spatial occurrence of MB axon pruning combined with mosaic analyses provide a platform to perform genetic screens and molecular dissections of these processes in unprecedented resolution. The MB is comprised of three types of neurons that are sequentially born from four identical neuroblasts per hemisphere. Out of the three MB neuronal types, only the γ neurons undergo axon pruning, indicating that the process is cell-type specific. During the larval stage, γ neurons project a bifurcated axon to the dorsal and medial lobes. At the onset of metamorphosis, the dendrites of the γ neurons as well as specific parts of the axons are eliminated by localized fragmentation in a process that peaks at about 18 hr after puparium formation. Subsequently, γ neurons undergo developmental axon regrowth, which is distinct from initial axon outgrowth, to occupy the adult specific lobe (Issman-Zecharya, 2014).
Axon pruning of MB γ neurons depends on the cell-autonomous expression of the nuclear steroid hormone receptor, ecdysone receptor B1 (EcR-B1). The expression of EcR-B1 is regulated by at least three distinct pathways: the cohesin complex, the TGF-β pathway, and a network of nuclear receptors comprised of ftz-f1 and Hr39. While expression of EcR-B1 is required for pruning, it is not sufficient to drive ectopic pruning either in γ neurons or in other MB neurons that do not undergo remodeling. This raises two possible nonmutually exclusive scenarios: (1) additional molecules are required to initiate pruning and (2) an inhibitory signal needs to be attenuated in the MB for pruning to occur. Additionally, the ubiquitin pathway is also cell-autonomously required in γ neurons for pruning, but the target that must be ubiquitinated remains unknown. Thus, while understanding of the cellular sequence of events culminating in the elimination of specific axonal branches is quite detailed, understanding of the molecular mechanisms remains incomplete (Issman-Zecharya, 2014).
In a forward genetic screen, this study identified a cell-autonomous role for the UV radiation resistance-associated gene (UVRAG) in MB γ neuron pruning. UVRAG was originally identified based on its ability to confer UV resistance to nucleotide excision repair deficient cells. It was later shown to function as a tumor suppressor gene deleted in various types of cancers including colon and gastric carcinomas (Ionov, 2004; Kim, 2008). UVRAG interacts with Atg6 (also known as Beclin1), another tumor suppressor gene, and together they promote autophagy in vitro (Liang, 2006). Their tumor suppression capabilities were first attributed to their autophagy-promoting function. However, a mutant form of UVRAG isolated from colon carcinomas promoted autophagy normally in cell culture. Both UVRAG and Atg6 are subunits in the phosphatidylinositol 3-kinase class III (PI3K-cIII) complex, involved in autophagy and endocytosis. Recent studies have found that UVRAG mediates endocytosis in an Atg6-dependent manner suggesting that as part of the PI3K-cIII complex, both proteins regulate various aspects of vesicle trafficking (Itakura, 2009; Thoresen, 2010). Two studies have recently identified new and seemingly unrelated functions for UVRAG in regulating DNA repair in response to UV-induced damage (Zhao, 2012) and ER to Golgi trafficking (He, 2014). Finally, an in vivo study has shown that UVRAG affects organ rotation in Drosophila by regulating Notch endocytosis in what seemed to be an Atg6-independent manner (Lee, 2011). A unifying understanding of the various aspects of UVRAG physiological function in vivo is still lacking. Likewise, although the PI3K-cIII complex has been extensively studied and implicated in autophagy, cytokinesis and endocytosis (Juhász, 2008; Thoresen, 2010), its physiological roles during the normal course of development are not known (Issman-Zecharya, 2014).
This study reports that UVRAG and the PI3K-cIII complex mediate the endosome-lysosome degradation of Ptc to promote axon pruning. Furthermore, the results suggest that Ptc represses pruning via a Smo- and Hh-independent manner. This study provides evidence for the existence of a pruning inhibitory pathway originating at the membrane of MB neurons (Issman-Zecharya, 2014).
This study shows that the endosomal-lysosomal pathway is cell-autonomously required for developmental axon pruning of mushroom body (MB) γ neurons. Genetic loss-of-function experiments indicate that UVRAG, a tumor suppressor gene previously linked to both endocytosis and autophagy, promotes pruning as part of the phosphatidylinositol 3-kinase class III (PI3K-cIII) complex and that UVRAG is required in MB neurons for the formation of phosphatidylinositol 3-phosphate (PI3P). The ESCRT-0 complex, which is recruited to the PI3 moiety on endosomal membranes, is required for pruning, indicating that endosome to multivesicular body maturation is critical for the normal progression of axon pruning and suggesting that it involves receptor downregulation. Genetic loss-of-function and gain-of-function experiments suggest that downregulation of the Hedgehog receptor Patched (Ptc) by the endocytic machinery is instrumental in promoting pruning. Finally, the results suggest that Ptc inhibits pruning in a smo-independent and likely also hh-independent manner (Issman-Zecharya, 2014).
A recent study suggested that UVRAG is required for Notch endocytosis during organ rotation in Drosophila in an Atg6-independent manner (Lee, 2011). While the current study shows that Atg6 is required for pruning, these seemingly contradicting results can be easily explained by specific allele differences. The Atg600096 allele, used in the previous study, is a P element insertion about 100 bp upstream of the Atg6 gene that does not necessarily create a null allele. Indeed, this study could also not see any effect of this allele on axon pruning. This study used an Atg61 null allele created by homologous recombination resulting in a strong effect on pruning. Furthermore, the data clearly show that the entire PI3K-cIII complex is required for axon pruning (Issman-Zecharya, 2014).
The PI3K-cIII complex has been implicated in a wide variety of membrane trafficking processes ranging from autophagy to endocytosis to cytokinesis (Thoresen, 2010). How the PI3K-cIII is regulated to participate in these different processes and its physiological roles in vivo are not well understood. While its role in promoting autophagy is supported by several studies (Burman, 2010 and Jaber, 2012), deleting the catalytic unit, Vps34, in sensory neurons does not affect autophagy, but rather endocytosis (Zhou, 2010). Whether this is a common feature of PI3K-cIII function in neurons remains to be further elucidated. One attractive hypothesis is that the PI3K-cIII function is determined by its complex composition. Indeed, it appears that in vitro, UVRAG and Atg14 are mutually exclusive subunits defining two distinct populations of the PI3K-cIII complex (Funderburk, 2010; Itakura, 2009). The current study is consistent with these findings, suggesting that UVRAG may define an endocytosis-specific PI3K-cIII complex at least in neurons. The full spectrum of the various PI3K-cIII complexes physiological roles in vivo remains to be further studied (Issman-Zecharya, 2014).
The PI3K-cIII complex phosphorylates PI to form PI3P on endosomal membranes. Indeed, this study found that UVRAG is essential for efficient PI3P formation and that PI3P is abundant throughout development. It is thus hypothesized that a PI3P binding protein mediates the effect of UVRAG and the PI3K-cIII complex on axon pruning. This study has identified Hrs, a subunit of the ESCRT-0 complex and a PI3P binding protein, as required for axon pruning. The role of ESCRT-0 in MVB maturation led to a hypothesis that the endolysosomal pathway is required to downregulate a signal that originates at the plasma membrane. While signaling can still occur in the early endosome, it is terminated at the MVB (Issman-Zecharya, 2014).
What is the identity of this transmembrane protein? Using genetic loss-of-function and gain-of-function experiments, it is suggested that Patched (Ptc) is at least one of the transmembrane proteins that is responsible for mediating the PI3K-cIII pruning defect. Strikingly, mutating ptc on the background of a Atg6 mutant significantly suppressed its pruning defect. Furthermore, overexpression of Ptc in WT brains resulted in a weak to mild pruning defect, depending on the Gal4 driver. Finally, overexpressing Ptc on the background of an endosomal defect significantly exacerbated the pruning defect. Together, these data suggest that Ptc mediates an inhibitory signal that needs to be attenuated for the normal progression of pruning. Interestingly, Ptc inactivation by endocytosis followed by lysosomal degradation was proposed before as a mechanism to activate the Hh pathway. What is the nature of this signal? Ptc is known to be the Hedgehog (Hh) receptor. Binding of Hh to Ptc relieves the Ptc-induced suppression of another transmembrane protein, Smoothened (Smo). Once derepressed, Smo initiates the intracellular Hh signal that culminates in the expression of specific nuclear transcription factors. Therefore this study tested the role of Smo and Hh in developmental axon pruning and, to surprisingly, demonstrated that both molecules seem to be irrelevant for pruning. Overexpressing Ptc mutant transgenes within MB neurons to identify the domains that are important for pruning inhibitions confirmed that Smo inhibition was not required to inhibit pruning. In contrast, the results suggest that the ligand binding domain is important. Because the results suggest that Hh is not required for pruning inhibition, it will be interesting to investigate in the future what other ligands might bind to Ptc. In this regard it is interesting to mention that a recent study has shown that Ptc is a lipoprotein receptor. The precise mechanism of Ptc action in MB neurons remains to be further elucidated in future studies (Issman-Zecharya, 2014).
This study has uncovered a role for the endocytic machinery in downregulating an inhibitory signal that is dependent on Ptc during MB axon pruning. A recently published study by Zhang (2014) has shown that the Rab5/ESCRT endocytic pathways are required to downregulate neuroglian (Nrg) to promote dendrite pruning of sensory neurons in Drosophila (Zhang, 2014). Both studies highlight that a combination of both promoting and inhibitory signals during developmental pruning is likely important to provide fail-safe mechanisms to regulate the process in a temporal, spatial, and cell-type specific resolution (Issman-Zecharya, 2014).
UV-induced DNA damage, a major risk factor for skin cancers, is primarily repaired by nucleotide excision repair (NER). UV radiation resistance-associated gene (UVRAG) is a tumor suppressor involved in autophagy. It was initially isolated as a cDNA partially complementing UV sensitivity in xeroderma pigmentosum (XP), but this was not explored further. This study show that UVRAG plays an integral role in UV-induced DNA damage repair. It localizes to photolesions and associates with DDB1 to promote the assembly and activity of the DDB2-DDB1-Cul4A-Roc1 (CRL4(DDB2)) ubiquitin ligase complex, leading to efficient XPC recruitment and global genomic NER. UVRAG depletion decreased substrate handover to XPC and conferred UV-damage hypersensitivity. The importance of UVRAG for UV-damage tolerance was confirmed using a Drosophila model. Furthermore, increased UV-signature mutations in melanoma correlate with reduced expression of UVRAG. These results identify UVRAG as a regulator of CRL4(DDB2)-mediated NER and suggest that its expression levels may influence melanoma predisposition (Yang, 2016).
UV radiation resistance-associated gene (UVRAG) is a tumor suppressor involved in autophagy, endocytosis and DNA damage repair, but how its loss contributes to colorectal cancer is poorly understood. This study shows that UVRAG deficiency in Drosophila intestinal stem cells leads to uncontrolled proliferation and impaired differentiation without preventing autophagy. As a result, affected animals suffer from gut dysfunction and short lifespan. Dysplasia upon loss of UVRAG is characterized by the accumulation of endocytosed ligands and sustained activation of STAT and JNK signaling, and attenuation of these pathways suppresses stem cell hyperproliferation. Importantly, the inhibition of early (dynamin-dependent) or late (Rab7-dependent) steps of endocytosis in intestinal stem cells also induces hyperproliferation and dysplasia. These data raise the possibility that endocytic, but not autophagic, defects contribute to UVRAG-deficient colorectal cancer development in humans (Nagy, 2016).
UVRAG encodes a homolog of yeast Vps38 in metazoans. UVRAG/Vps38 and Atg14 are mutually exclusive subunits of two different Vps34 lipid kinase complexes, both of which contain Vps34, Vps15 and Atg6/Beclin 1 (Itakura, 2008). It is well established that Vps38 is required for endosome maturation and vacuolar and lysosomal protein sorting, whereas Atg14 is specific for autophagy in yeast. However, the function of UVRAG is much more controversial in mammalian cells. Although UVRAG was originally found to have dual roles in autophagy through promotion of autophagosome formation and fusion with lysosomes in various cultured cell lines based on, predominantly, overexpression experiments, recent reports have described that autophagosomes are normally generated and fused with lysosomes in the absence of UVRAG in cultured mammalian (HeLa) cells and in the Drosophila fat body (Jiang, 2014; Takáts, 2014; Nagy, 2016 and references therein).
The discoveries of UVRAG mutations in colorectal cancer cells, and that its overexpression increases autophagy and suppresses the proliferation of certain cancer cell lines, altogether suggest that this gene functions as an autophagic tumor suppressor. Such a role for UVRAG is thought to be related to its binding to Beclin 1, a haploinsufficient tumor suppressor gene required for autophagy. UVRAG appears to play roles similar to yeast Vps38 in the Drosophila fat body, and developing eye and wing: its loss leads to the accumulation of multiple endocytic receptors and ligands in an endosomal compartment, impaired trafficking of Lamp1 and Cathepsin L to the lysosome, and defects in the biogenesis of lysosome-related pigment granules. However, whether this gene is also required for the maintenance of intestinal homeostasis in Drosophila was unclear because the loss of UVRAG did not lead to uncontrolled cell proliferation in the developing eye or wing according to these reports. The current results showing that Uvrag deficiency causes intestinal dysplasia suggest that this gene is also important for the proper functioning of the adult gut in Drosophila (Nagy, 2016).
A surprising aspect of this work is that UVRAG appears to function independently of autophagy in the intestine. There are other lines of evidence that also support that UVRAG has a more important role in endocytic maturation than in autophagy. First, it has been shown that truncating mutations in UVRAG that are associated with microsatellite-unstable colon cancer cell lines do not disrupt autophagy. Second, UVRAG depletion in HeLa cells does not prevent the formation or fusion of autophagosomes with lysosomes, but it does interfere with Egfr degradation (Jiang, 2014). Third, a very recent paper has shown that overexpression of the colorectal-cancer-associated truncated form of UVRAG promotes tumorigenesis independently of autophagy status, that is, both in control and Atg5-knockout cells (He, 2015). That paper, again, relied on the overexpression of full-length or truncated forms of UVRAG, rather than the analysis of cancer-related mutations of the endogenous locus. Fourth, the endocytic function of UVRAG has been found to be required for developmental axon pruning that is independent of autophagy in Drosophila (Issman-Zecharya, 2014; Nagy, 2016 and references therein).
The results of this study indicate that UVRAG loss is accompanied with the sustained activation of JNK and STAT signaling in ISCs and EBs, and that these pathways are required for dysplasia in this setting. Sustained activation of these signaling routes is likely to be connected to the disruption of endocytic flux in the absence of UVRAG, because inhibiting endocytic uptake or degradation through dominant-negative dynamin expression or RNAi of Rab7, respectively, also leads to intestinal dysplasia. It is worth noting that the effects of inhibiting Shibire/dynamin function led to a much more severe hyperproliferation of ISCs and early death of animals. In line with this, the loss of early endocytic regulators, such as Rab5, in the developing eye causes overproliferation of cells and lethality during metamorphosis. Although eye development is not perturbed by the loss of the late endocytic regulators UVRAG or Rab7, these proteins are clearly important for controlling ISC proliferation and differentiation (Nagy, 2016).
A recent paper shows that hundreds of RNAi lines cause the expansion of the esg-GFP compartment in 1-week-old animals, which might be due to an unspecific ISC stress response in some cases. However, several lines of evidence support that impaired UVRAG-dependent endocytic degradation is specifically required to prevent intestinal dysplasia. First of all, activation of JNK stress signaling in esg-GFP-positive cells induces short-term ISC proliferatio, and almost all stem cells are lost through apoptosis by the 2- to 3-week age, the time when the Uvrag-mutant phenotype becomes obvious. In fact, UVRAG loss resembles an early-onset age-associated dysplasia that is normally observed in old (30-60 days) flies and involves the simultaneous activation of both JNK and STAT signaling. Second, UVRAG RNAi in ISCs and EBs leads to paracrine activation of the cytokine Unpaired3 in enterocytes, one of the hallmarks of niche appropriation by Notch-negative tumors. However, autocrine expression of the Unpaired proteins and JNK activation is observed in Uvrag-knockdown cells, unlike in Notch-negative tumors, and EBs with active Notch signaling accumulate in the absence of UVRAG, so the two phenotypes are clearly different. Third, it is the loss of autophagy that could be expected to mimic a stress response and perhaps induce stem cell tumors, but this does not seem to be the case – ISCs with Atg5 or Atg14 RNAi proliferate less in 3-week-old animals and an overall decrease of the esg-GFP compartment is seen, as opposed to the Uvrag-deletion phenotype (Nagy, 2016).
Taken together, this work indicates that endocytic maturation and degradation serves to prevent early-onset intestinal dysplasia in Drosophila, and its deregulation could be relevant for the development of colorectal cancer in humans (Nagy, 2016).
Membrane fusion is generally controlled by Rabs, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), and tethering complexes. Syntaxin 17 (STX17) was recently identified as the autophagosomal SNARE required for autophagosome-lysosome fusion in mammals and Drosophila. To better understand the mechanism of autophagosome-lysosome fusion, this study sought STX17-interacting proteins. Immunoprecipitation and mass spectrometry analysis identified vacuolar protein sorting 33A (VPS33A) and VPS16, which are components of the homotypic fusion and protein sorting (HOPS)-tethering complex. It was further confirmed that all HOPS components were coprecipitated with STX17. Knockdown of VPS33A, VPS16, or VPS39 blocked autophagic flux and caused accumulation of STX17- and microtubule-associated protein light chain (LC3)-positive autophagosomes. The endocytic pathway was also affected by knockdown of VPS33A, as previously reported, but not by knockdown of STX17. By contrast, ultraviolet irradiation resistance-associated gene (UVRAG), a known HOPS-interacting protein, did not interact with the STX17-HOPS complex and may not be directly involved in autophagosome-lysosome fusion. Collectively these results suggest that, in addition to its well-established function in the endocytic pathway, HOPS promotes autophagosome-lysosome fusion (Jiang, 2014)
Atg6 (Beclin 1 in mammals) is a core component of the Vps34 PI3K (III) complex, which promotes multiple vesicle trafficking pathways. Atg6 and Vps34 form two distinct PI3K (III) complexes in yeast and mammalian cells, either with Atg14 or with UVRAG. The functions of these two complexes are not entirely clear, as both Atg14 and UVRAG have been suggested to regulate both endocytosis and autophagy. In this study, a microscopic analysis of UVRAG, Atg14, or Atg6 loss-of-function cells was performed in the developing Drosophila wing. Both autophagy and endocytosis are seriously impaired and defective endolysosomes accumulate upon loss of Atg6. Atg6 is required for the downregulation of Notch and Wingless signaling pathways; thus it is essential for normal wing development. Moreover, the loss of Atg6 impairs cell polarity. Atg14 depletion results in autophagy defects with no effect on endocytosis or cell polarity, while the silencing of UVRAG phenocopies all but the autophagy defect of Atg6 depleted cells. Thus, these results indicate that the UVRAG-containing PI3K (III) complex is required for receptor downregulation through endolysosomal degradation and for the establishment of proper cell polarity in the developing wing, while the Atg14-containing complex is involved in autophagosome formation (Lorincz, 2014).
Homotypic fusion and vacuole protein sorting (HOPS) is a tethering complex required for trafficking to the vacuole/lysosome in yeast. Specific interaction of HOPS with certain SNARE (soluble NSF attachment protein receptor) proteins ensures the fusion of appropriate vesicles. HOPS function is less well characterized in metazoans. This study shows show that all six HOPS subunits (Vps11 [vacuolar protein sorting 11]/CG32350, Vps18/Dor, Vps16A, Vps33A/Car, Vps39/CG7146, and Vps41/Lt) are required for fusion of autophagosomes with lysosomes in Drosophila. Loss of these genes results in large-scale accumulation of autophagosomes and blocks autophagic degradation under basal, starvation-induced, and developmental conditions. HOPS colocalizes and interacts with Syntaxin 17 (Syx17), the recently identified autophagosomal SNARE required for fusion in Drosophila and mammals, suggesting their association is critical during tethering and fusion of autophagosomes with lysosomes. HOPS, but not Syx17, is also required for endocytic down-regulation of Notch and Boss in developing eyes and for proper trafficking to lysosomes and eye pigment granules. This study also showed that the formation of autophagosomes and their fusion with lysosomes is largely unaffected in null mutants of Vps38/UVRAG (UV radiation resistance associated), a suggested binding partner of HOPS in mammals, while endocytic breakdown and lysosome biogenesis is perturbed. These results establish the role of HOPS and its likely mechanism of action during autophagy in metazoans (Takats, 2014).
Heterotaxy characterized by abnormal left-right body asymmetry causes diverse congenital anomalies. Organ rotation is a crucial developmental process to establish the left-right patterning during animal development. However, the molecular basis of how organ rotation is regulated is poorly understood. This study reports that Drosophila UV-resistance associated gene (UVRAG), a tumor suppressor that regulates autophagy and endocytosis, plays unexpected roles in controlling organ rotation. Loss-of-function mutants of UVRAG show seriously impaired organ rotation phenotypes, which are associated with defects in endocytic trafficking rather than autophagy. Blunted endocytic degradation by UVRAG deficiency causes endosomal accumulation of Notch, resulting in abnormally enhanced Notch activity. Knockdown of Notch itself or expression of a dominant negative form of Notch transcriptional co-activator Mastermind is sufficient to rescue the rotation defect in UVRAG mutants. Consistently, UVRAG-mutated heterotaxy patient cells also display highly increased Notch protein levels. These results suggest evolutionarily conserved roles of UVRAG in organ rotation by regulating Notch endocytic degradation (Lee, 2011).
Degradation of cytoplasmic components by autophagy requires the class III phosphatidylinositol 3 [PI3]-kinase Vps34, but the mechanisms by which this kinase and its lipid product PI3 phosphate (PI3P) promote autophagy are unclear. In mammalian cells, Vps34, with the proautophagic tumor suppressors Beclin1/Atg6, Bif-1, and UVRAG, forms a multiprotein complex that initiates autophagosome formation. Distinct Vps34 complexes also regulate endocytic processes that are critical for late-stage autophagosome-lysosome fusion. In contrast, Vps34 may also transduce activating nutrient signals to mammalian target of rapamycin (TOR), a negative regulator of autophagy. To determine potential in vivo functions of Vps34, mutations were generated in the single Drosophila Vps34 orthologue (Phosphotidylinositol 3 kinase 59F), causing cell-autonomous disruption of autophagosome/autolysosome formation in larval fat body cells. Endocytosis is also disrupted in Vps34-/- animals, but this does not account for their autophagy defect. Unexpectedly, TOR signaling is unaffected in Vps34 mutants, indicating that Vps34 does not act upstream of TOR in this system. Instead, TOR/Atg1 signaling regulates the starvation-induced recruitment of PI3P to nascent autophagosomes. These results suggest that Vps34 is regulated by TOR-dependent nutrient signals directly at sites of autophagosome formation (Juhász, 2008).
Engulfment of cytoplasmic material into specialized double-membrane vesicles known as autophagosomes is the defining feature of a process referred to as macroautophagy or simply autophagy. Subsequent fusion of autophagosomes with the endolysosomal network leads to hydrolytic degradation of the sequestered material. This process provides eukaryotic cells with a mechanism for cytoplasmic renewal by which they can rid themselves of defective organelles and protein complexes. In addition, nonselective autophagy can be induced to high levels by starvation, providing an internal source of nutrients on which cells can survive extended periods of nutrient deprivation. Conversely, under some circumstances autophagy may be used as a killing mechanism, acting as an alternative or augmentation to apoptotic cell death. As autophagy has been implicated in several physiological and pathological conditions, including neurodegeneration, tumorigenesis, and aging, better understanding of the molecular mechanisms controlling autophagy and identification of pharmacological regulators of this process are important goals (Juhász, 2008).
Wortmannin and 3-methyladenine are well established inhibitors of autophagy. These compounds are broad-spectrum phosphatidylinositol 3 [PI3]-kinase inhibitors that disrupt autophagy by inhibiting Vps34, the enzymatic component of a multiprotein complex which also includes Vps15, Beclin1/Atg6, UVRAG, and Bif-1 in mammals and Vps15, Atg6, and Atg14 in yeast. Localized production of PI3 phosphate (PI3P) by Vps34 can act to recruit proteins containing FYVE and PX domains to specific membrane compartments . In yeast, this Vps34 complex is critical for recruiting autophagy-related (Atg) proteins to the preautophagosomal structure, the yeast-specific site of autophagosome formation. The role of PI3P in autophagosome biogenesis is less well understood in higher eukaryotes, and whether it functions at the autophagosomal, the donor, or another membrane has not been determined (Juhász, 2008).
Vps34 is also required more broadly for several vesicular trafficking processes that may have indirect impacts on autophagy. These include sorting of hydrolytic enzymes to the lysosome/vacuole and early steps in the endocytic pathway. In mammalian cells, autophagosomes have been shown to fuse with early or late endosomes before fusion with lysosomes, resulting in intermediate structures known as amphisomes. Recently, mutations in components of the endosomal sorting complex required for transport (ESCRT) complex, which is required for the transition from early to late (multivesicular) endosomes, have been shown to block autophagy by inhibiting autophagosome-endosome fusion. Thus, the effect of PI3-kinase inhibitors on autophagy may be due, in part, to these more general trafficking functions of Vps34 (Juhász, 2008).
Recent work has shown that Vps34 can also function in a nutrient-sensing pathway upstream of the target of rapamycin (TOR) in several mammalian cell lines. Disruption of Vps34 activity with blocking antibodies or siRNA was found to inhibit activation of TOR by insulin, amino acids, and glucose. As TOR signaling inhibits autophagy, these findings are at odds with the conserved role of Vps34 in promoting autophagy under starvation conditions, suggesting that distinct complexes or pools of Vps34 may be subject to different modes of regulation (Juhász, 2008).
This paper addresses how these multiple potential roles of Vps34 are coordinated to regulate autophagy in Drosophila. The findings suggest that despite a critical role for Vps34 in endocytic uptake and recycling, its primary function in autophagy in vivo is limited to its direct role at the nascent autophagosome. Vps34 is not required for TOR activity in this system, and starvation results in a TOR/Atg1-dependent recruitment of Vps34 activity to the autophagosomal membrane (Juhász, 2008).
Previous work in mammalian and yeast systems has identified a wide range of vesicle trafficking processes regulated by Vps34, including autophagy, endocytosis, endosome maturation, and both anterograde and retrograde trafficking between the Golgi and lysosome. The current findings indicate that despite these activities, the in vivo role of Vps34 in autophagy is largely limited to its function at the autophagosome. Although fluid-phase endocytosis, endocytic recycling of Notch, and trafficking of lysosomal proteins are disrupted by mutation of Vps34, the results suggest that events subsequent to autophagosome formation, including fusion between autophagosomes and endosomes or lysosomes and subsequent lysosomal degradation, are not rate limiting in the absence of Vps34. Why does endocytic disruption lead to autophagosome fusion defects in ESCRT mutants but not in Vps34 mutants? Accumulation of endocytic tracer at the periphery of Vps34 mutant cells suggests that Vps34 functions at an early step of endocytosis, and apparently this event, as well as normal endocytic flux is not essential for fusion of autophagosomes with elements of the endosomal-lysosomal compartment. Interestingly, the accumulation of autophagosomes in ESCRT/Vps34 double mutants indicates that loss of Vps34 does not completely prevent autophagosome formation. Similarly, the lack of autophagosome accumulation in Vps34 single mutants indicates that ESCRT complexes are at least partially functional in the absence of Vps34. Thus, PI3P may not be absolutely essential for these processes, or perhaps sufficient levels of PI3P are generated independently of Vps34 by the class II PI3 kinase or by PI(3,4)P or PI(3,5)P phosphatases. Since ESCRT components are required for multivesicular body formation but not autophagy in yeast, it will be interesting to determine whether the requirement for ESCRT complexes in autophagy in higher eukaryotes reflects their role in multivesicular body formation or an alternate function (Juhász, 2008).
The cellular compartment in which Vps34 acts to promote autophagy and the mechanisms by which it is regulated by nutrient signals have remained unresolved. In mammalian cells, Beclin1/Atg6 has been reported to localize to the trans-Golgi network, ER, and mitochondria. It was recently shown that Beclin1-Vps34 complexes can be inhibited by the antiapoptotic factor Bcl-2 in a nutrient-dependent manner. Bcl-2 mutants that are targeted to the ER, but not to mitochondria, retain their capacity to inhibit starvation-induced autophagy, suggesting that the ER is an important site of Beclin1-Vps34 regulation. However, it is unknown how these organelles contribute to the formation of autophagosomes, and recent studies suggest that rather than budding off a preexisting compartment, the autophagosomal membrane is likely to form de novo from small lipid transport vesicles or lipoprotein complexes. The finding that myc-2xFYVE is recruited to GFP-Atg8a-positive structures under starvation conditions indicates that Vps34 activity is targeted directly to autophagosomes in a TOR/Atg1-dependent manner. Although these results do not distinguish between a role for TOR/Atg1 signaling in regulating Vps34 activity versus providing a platform on which Vps34 complexes can assemble, together with these previous studies they indicate that Vps34 is likely to promote autophagy by different mechanisms from multiple cellular locations (Juhász, 2008).
How the TOR signaling pathway senses intracellular levels of nutrients, such as amino acids, has been poorly understood despite considerable work in yeast, mammalian, and other model systems. The recent identification of Vps34 as a transducer of this signal in mammalian cells thus represents a significant new insight into this issue. However, further work is necessary to determine the extent to which this mechanism is generally conserved, since starvation appears to have opposing effects on Vps34 activity in different cell types. The results presented in this study fail to support this model in Drosophila; mutation of Vps34 does not appear to influence TOR-dependent phenotypes nor to disrupt TOR-dependent signaling. This may reflect a fundamental difference in signaling mechanisms between the fly and mammalian systems. The makeup of Vps34 complexes has diverged significantly between yeast and metazoans, and perhaps components of this complex, such as Ambra1, that appear to be unique to vertebrates may confer functions not found in flies. It is also possible that production of PI3P by Vps34-independent mechanisms is more efficient in D. melanogaster than in mammalian cells and, thus, a role for Vps34 in TOR signaling may be obscured by these other sources. The continued ESCRT function and basal level of autophagy in Vps34 null mutants are consistent with this possibility. Alternatively, the current findings may reflect important differences between the roles of TOR and Vps34 in vivo versus in cultured cells as well as the experimental paradigms of these systems. For example, although complete starvation is commonly used to inactivate TOR in cell culture studies, such experiments may not accurately mimic physiologically relevant events, given the inherent capacity of intact organisms to buffer changes in nutrient levels. Additional studies in an in vivo mammalian system will be helpful to clarify these issues (Juhász, 2008).
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Burman, C. and Ktistakis, N. T. (2010). Regulation of autophagy by phosphatidylinositol 3-phosphate. FEBS Lett 584: 1302-1312. PubMed ID: 20074568
Funderburk, S. F., Wang, Q. J. and Yue, Z. (2010). The Beclin 1-VPS34 complex--at the crossroads of autophagy and beyond. Trends Cell Biol 20: 355-362. PubMed ID: 20356743
He, S., O'Connell, D., Zhang, X., Yang, Y. and Liang, C. (2014). The intersection of Golgi-ER retrograde and autophagic trafficking. Autophagy 10: 180-181. PubMed ID: 24246972
He, S., Zhao, Z., Yang, Y., O'Connell, D., Zhang, X., Oh, S., Ma, B., Lee, J. H., Zhang, T., Varghese, B., Yip, J., Dolatshahi Pirooz, S., Li, M., Zhang, Y., Li, G. M., Ellen Martin, S., Machida, K. and Liang, C. (2015). Truncating mutation in the autophagy gene UVRAG confers oncogenic properties and chemosensitivity in colorectal cancers. Nat Commun 6: 7839. PubMed ID: 26234763
Ionov, Y., Nowak, N., Perucho, M., Markowitz, S. and Cowell, J. K. (2004). Manipulation of nonsense mediated decay identifies gene mutations in colon cancer Cells with microsatellite instability. Oncogene 23: 639-645. PubMed ID: 14737099
Issman-Zecharya, N. and Schuldiner, O. (2014). The PI3K class III complex promotes axon pruning by downregulating a Ptc-derived signal via endosome-lysosomal degradation. Dev Cell 31: 461-473. PubMed ID: 25458013
Itakura, E., Kishi, C., Inoue, K. and Mizushima, N. (2008). Beclin 1 forms two distinct phosphatidylinositol 3-kinase complexes with mammalian Atg14 and UVRAG. Mol Biol Cell 19: 5360-5372. PubMed ID: 18843052
Itakura, E. and Mizushima, N. (2009). Atg14 and UVRAG: mutually exclusive subunits of mammalian Beclin 1-PI3K complexes. Autophagy 5: 534-536. PubMed ID: 19223761
Jaber, N., Dou, Z., Chen, J. S., Catanzaro, J., Jiang, Y. P., Ballou, L. M., Selinger, E., Ouyang, X., Lin, R. Z., Zhang, J. and Zong, W. X. (2012). Class III PI3K Vps34 plays an essential role in autophagy and in heart and liver function. Proc Natl Acad Sci U S A 109: 2003-2008. PubMed ID: 22308354
Jiang, P., Nishimura, T., Sakamaki, Y., Itakura, E., Hatta, T., Natsume, T. and Mizushima, N. (2014). The HOPS complex mediates autophagosome-lysosome fusion through interaction with syntaxin 17. Mol Biol Cell 25: 1327-1337. PubMed ID: 24554770
Juhasz, G., Hill, J. H., Yan, Y., Sass, M., Baehrecke, E. H., Backer, J. M. and Neufeld, T. P. (2008). The class III PI3K Vps34 promotes autophagy and endocytosis but not TOR signaling in Drosophila. J Cell Biol 181: 655-666. PubMed ID: 18474623
Kim, M. S., Jeong, E. G., Ahn, C. H., Kim, S. S., Lee, S. H. and Yoo, N. J. (2008). Frameshift mutation of UVRAG, an autophagy-related gene, in gastric carcinomas with microsatellite instability. Hum Pathol 39: 1059-1063. PubMed ID: 18495205
Lee, G., Liang, C., Park, G., Jang, C., Jung, J. U. and Chung, J. (2011). UVRAG is required for organ rotation by regulating Notch endocytosis in Drosophila. Dev Biol 356: 588-597. PubMed ID: 21729695
Liang, C., Feng, P., Ku, B., Dotan, I., Canaani, D., Oh, B. H. and Jung, J. U. (2006). Autophagic and tumour suppressor activity of a novel Beclin1-binding protein UVRAG. Nat Cell Biol 8: 688-699. PubMed ID: 16799551
Lorincz, P., Lakatos, Z., Maruzs, T., Szatmari, Z., Kis, V. and Sass, M. (2014). Atg6/UVRAG/Vps34-containing lipid kinase complex is required for receptor downregulation through endolysosomal degradation and epithelial polarity during Drosophila wing development. Biomed Res Int 2014: 851349. PubMed ID: 25006588
Nagy, P., Kovacs, L., Sandor, G. O. and Juhasz, G. (2016). Stem-cell-specific endocytic degradation defects lead to intestinal dysplasia in Drosophila. Dis Model Mech 9: 501-512. PubMed ID: 26921396
Takats, S., Pircs, K., Nagy, P., Varga, A., Karpati, M., Hegedus, K., Kramer, H., Kovacs, A. L., Sass, M. and Juhasz, G. (2014). Interaction of the HOPS complex with Syntaxin 17 mediates autophagosome clearance in Drosophila. Mol Biol Cell 25: 1338-1354. PubMed ID: 24554766
Thoresen, S. B., Pedersen, N. M., Liestol, K. and Stenmark, H. (2010). A phosphatidylinositol 3-kinase class III sub-complex containing VPS15, VPS34, Beclin 1, UVRAG and BIF-1 regulates cytokinesis and degradative endocytic traffic. Exp Cell Res 316: 3368-3378. PubMed ID: 20643123
Yang, Y., He, S., Wang, Q., Li, F., Kwak, M. J., Chen, S., O'Connell, D., Zhang, T., Pirooz, S. D., Jeon, Y., Chimge, N. O., Frenkel, B., Choi, Y., Aldrovandi, G. M., Oh, B. H., Yuan, Z. and Liang, C. (2016). Autophagic UVRAG promotes UV-induced photolesion repair by activation of the CRL4(DDB2) E3 ligase. Mol Cell 62: 507-519. PubMed ID: 27203177
Zhang, H., Wang, Y., Wong, J. J., Lim, K. L., Liou, Y. C., Wang, H. and Yu, F. (2014). Endocytic pathways downregulate the L1-type cell adhesion molecule neuroglian to promote dendrite pruning in Drosophila. Dev Cell 30: 463-478. PubMed ID: 25158855
Zhou, X., Wang, L., Hasegawa, H., Amin, P., Han, B. X., Kaneko, S., He, Y. and Wang, F. (2010). Deletion of PIK3C3/Vps34 in sensory neurons causes rapid neurodegeneration by disrupting the endosomal but not the autophagic pathway. Proc Natl Acad Sci U S A 107: 9424-9429. PubMed ID: 20439739
date revised: 18 September 2016
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