InteractiveFly: GeneBrief

Vacuolar H+ ATPase subunit 68-2: Biological Overview | References

Gene name - Vacuolar H+ ATPase subunit 68-2

Synonyms -

Cytological map position- 34A3-34A3

Function - enzyme

Keywords - vacuolar proton pump, regulation of pH, Vesicles Notch pathway, acidification of the endosomal compartment, Malpighian tubules

Symbol - Vha68-2

FlyBase ID: FBgn0263598

Genetic map position - 2L:12,974,322..12,978,615 [+]

Classification - V-ATPase_V1_A: V-type (H+)-ATPase V1, A subunit

Cellular location - vesicular

NCBI links: EntrezGene

Vha68-2 orthologs: Biolitmine
Recent literature
Overend, G., Luo, Y., Henderson, L., Douglas, A.E., Davies, S.A. and Dow, J.A. (2016). Molecular mechanism and functional significance of acid generation in the Drosophila midgut. Sci Rep 6: 27242. PubMed ID: 27250760
The gut of Drosophila melanogaster includes a proximal acidic region (~pH 2), however the genome lacks the H+/K+ ATPase characteristic of the mammalian gastric parietal cell, and the molecular mechanisms of acid generation are poorly understood. This study shows that maintenance of the low pH of the acidic region is dependent on H+ V-ATPase, together with carbonic anhydrase and five further transporters or channels that mediate K+, Cl- and HCO3- transport. Abrogation of the low pH does not influence larval survival under standard laboratory conditions, but is deleterious for insects subjected to high Na+ or K+ load. Insects with elevated pH in the acidic region display increased susceptibility to Pseudomonas pathogens and increased abundance of key members of the gut microbiota (Acetobacter and Lactobacillus), suggesting that the acidic region has bacteriostatic or bacteriocidal activity. Conversely, the pH of the acidic region is significantly reduced in germ-free Drosophila, indicative of a role of the gut bacteria in shaping the pH conditions of the gut. These results demonstrate that the acidic gut region protects the insect and gut microbiome from pathological disruption, and shed light on the mechanisms by which low pH can be maintained in the absence of H+, K+ ATPase. 

Wissel, S., Harzer, H., Bonnay, F., Burkard, T. R., Neumuller, R. A. and Knoblich, J. A. (2018). Time-resolved transcriptomics in neural stem cells identifies a v-ATPase/Notch regulatory loop. J Cell Biol. PubMed ID: 29959232
Drosophila melanogaster neural stem cells (neuroblasts [NBs]) divide asymmetrically by differentially segregating protein determinants into their daughter cells. Although the machinery for asymmetric protein segregation is well understood, the events that reprogram one of the two daughter cells toward terminal differentiation are less clear. This study used time-resolved transcriptional profiling to identify the earliest transcriptional differences between the daughter cells on their way toward distinct fates. By screening for coregulated protein complexes, vacuolar-type H(+)-ATPase (v-ATPase) was identified among the first and most significantly down-regulated complexes in differentiating daughter cells. v-ATPase is essential for NB growth and persistent activity of the Notch signaling pathway. The data suggest that v-ATPase and Notch form a regulatory loop that acts in multiple stem cell lineages both during nervous system development and in the adult gut. This study provides a unique resource for investigating neural stem cell biology and demonstrate that cell fate changes can be induced by transcriptional regulation of basic, cell-essential pathways.
Lim, D. H., Lee, S., Choi, M. S., Han, J. Y., Seong, Y., Na, D., Kwon, Y. S. and Lee, Y. S. (2020). The conserved microRNA miR-8-3p coordinates the expression of V-ATPase subunits to regulate ecdysone biosynthesis for Drosophila metamorphosis. FASEB J. PubMed ID: 32196731
The steroid hormone ecdysone is the central regulator of insect metamorphosis, during which a growing, immature larva is remodeled, through pupal stages, to a reproductive adult. However, the underlying mechanisms of ecdysone-mediated metamorphosis remain to be fully elucidated. This study identified metamorphosis-associated microRNAs (miRNAs) and their potential targets by cross-linking immunoprecipitation coupled with deep sequencing of endogenous Argonaute 1 protein in Drosophila. Interestingly, miR-8-3p targeted five Vha genes encoding distinct subunits of vacuolar H(+) -ATPase (V-ATPase; see Vha68-2), which has a vital role in the organellar acidification. The expression of ecdysone-responsive miR-8-3p is normally downregulated during Drosophila metamorphosis, but temporary overexpression of miR-8-3p in the whole body at the end of larval development led to defects in metamorphosis and survival, hallmarks of aberrant ecdysone signaling. In addition, miR-8-3p was expressed in the prothoracic gland (PG), which produces and releases ecdysone in response to prothoracicotropic hormone (PTTH). Notably, overexpression of miR-8-3p or knockdown of its Vha targets in the PG resulted in larger than normal, ecdysone-deficient larvae that failed to develop into the pupal stage but could be rescued by ecdysone feeding. Moreover, these animals showed defective PTTH signaling with a concomitant decrease in the expression of ecdysone biosynthetic genes. Tnis study also demonstrated that the regulatory network between the conserved miR-8-3p/miR-200 family and V-ATPase was functional in human cells. Consequently, these data indicate that the coordinated regulation of V-ATPase subunits by miR-8-3p is involved in Drosophila metamorphosis by controlling the ecdysone biosynthesis.


Evidence indicates that endosomal entry promotes signaling by the Notch receptor, but the mechanisms involved are not clear. In a search for factors that regulate Notch activation in endosomes, mutants were isolated in Drosophila genes that encode subunits of the vacuolar ATPase (V-ATPase) proton pump. Cells lacking V-ATPase function display impaired acidification of the endosomal compartment and a correlated failure to degrade endocytic cargoes. V-ATPase mutant cells internalize Notch and accumulate it in the lysosome, but surprisingly also show a substantial loss of both physiological and ectopic Notch activation in endosomes. V-ATPase activity is required in signal-receiving cells for Notch signaling downstream of ligand activation but upstream of γ-secretase-dependent S3 cleavage. These data indicate that V-ATPase, probably via acidification of early endosomes, promotes not only the degradation of Notch in the lysosome but also the activation of Notch signaling in endosomes. The results also suggest that the ionic properties of the endosomal lumen might regulate Notch cleavage, providing a rationale for physiological as well as pathological endocytic control of Notch activity (Vaccari, 2010).

Cell-cell signaling via the Notch receptor is used throughout development to regulate multiple cell behaviors, and inappropriate activation of Notch is emerging as a common hallmark of an increasing number of cancers. Thus, resolving the mechanisms by which Notch signaling is regulated is of great importance and of widespread interest in order to understand human development as well as to devise effective anticancer therapies. In response to ligand engagement, the Notch receptor is activated by S3 cleavage, a γ-secretase-mediated intramembrane proteolysis that liberates the Notch intracellular domain from its transmembrane anchor, allowing the soluble form to travel to the nucleus, where it regulates the transcription of a variety of important target genes (Vaccari, 2010).

Mounting evidence, particularly in Drosophila, has pointed to the unexpected involvement of the endosomal system in regulating activation of the Notch receptor in signal-receiving cells. Importantly, endosomal regulators can either increase or decrease Notch signaling depending on the specific site of the endocytic pathway at which they act. For example, factors that positively regulate traffic from the cell surface to endosomes, including Dynamin (Shibire -- FlyBase), the ubiquitin ligase Deltex, the syntaxin Avalanche (Avl; Syntaxin 7), the GTPase Rab5, the Rab5 effector Rabenosyn-5, and the Sec1/Munc18 family protein Vps45, are required to promote signaling. By contrast, Endosomal sorting required for transport (ESCRT) complex components, the C2-domain protein Lethal (2) giant discs 1 (Lgd), and the ubiquitin ligase Su(dx), which subsequently sort cargo within the endosome towards lysosomal degradation, are required to prevent excess signaling. Much of this evidence points to early endosomes as important sites of signaling activation. However, the molecules and mechanisms that restrict activation of the Notch receptor to this endosomal compartment remain unknown (Vaccari, 2010).

To identify genes involved in endocytic control of the Notch receptor, a collection of Drosophila mutants enriched for endocytic regulators (Menut, 2007) was screened to identify those that display altered Notch localization. Eye imaginal discs consisting predominantly of mutant cells were generated and immunostained using Notch antibodies. It was found that discs mutant for the single allele complementation group MENE2L-R6 accumulate high levels of Notch as compared with wild-type (WT) discs. Clones of MENE2L-R6 mutant cells generated in the context of a mosaic eye disc also displayed substantial accumulation of Notch, which was found in large intracellular puncta. MENE2L-R6 discs were consistently smaller than WT discs and also displayed aberrant morphology, compromised epithelial polarity, and upregulated Matrix metalloprotease 1 (Mmp1) production. Together, these data suggest that R6 disrupts a gene that regulates Notch trafficking as well as the proper growth and organization of imaginal disc tissue (Vaccari, 2010).

The R6 mutant by complementation was mapped to a deficiency that removes the chromosomal region 34A3. Tests with lethal transposon insertions within the deficiency region revealed that R6 fails to complement P[PZ]l(2)01510, which is inserted in the first intron of the predicted gene CG3762. CG3762 is also known as Vha68-2, the most widely expressed of the three Drosophila genes that encode the A subunit of the multiprotein vacuolar ATPase (V-ATPase) proton pump (Allan, 2005). The A and B subunits form a hexamer that binds nucleotide (ATP) in the peripheral V1 sector; nucleotide binding and hydrolysis are essential for transmembrane proton conductance through the integral membrane V0 sector. Sequencing of Vha68-2 in R6 mutants revealed a nonsense mutation in the region encoding the C-terminal domain. Based on the crystal structure of the related A-ATPase subunit A (Maegawa, 2006), the truncated C-terminal domain of the R6 mutant protein, even if expressed, is likely to lack part of the region that contacts subunits D and F, which are involved in torque generation. Since torque generation is essential for proton transport and thus endomembrane acidification, the ability of Vha68-2 mutant cells to incorporate the vital dye Lysotracker, which accumulates in acidified lysosomes, was tested. Compared with WT cells or cells mutant for the endosomal component Hrs, it was found that Vha68-2 mutant cells incorporate very low levels of Lysotracker, indicating impairment of the ability to acidify endocytic organelles (Vaccari, 2010).

Failure of Lysotracker incorporation into Vha68-2 mutant cells is not due to absent endocytic structures, as staining with endosomal markers and electron microscopy revealed that both early and late endosomes, as well as lysosomes, are present in mutant cells. In addition, ultrastructural analysis of the morphology of the endocytic compartment revealed that Vha68-2 mutant cells contain, compared with WT, more multivesicular bodies (MVBs), a degradative endosomal organelle. These MVBs were also enlarged and contained a high number of internal luminal vesicles (ILVs). Consistent with a role for acidification in activating low-pH lipid hydrolases, these data suggest that Vha68-2 is required for lysosomal degradation of ILVs. Overall, the Lysotracker incorporation and the ultrastructural analyses indicate that Vha68-2 causes a substantial loss of proton transport activity (Vaccari, 2010).

Recent evidence suggests that the route and rate of Notch traffic through endocytic compartments can be regulated to either potentiate or diminish signaling activity. The results of this study identify a physiological mechanism to account for this effect on signaling. A set of genes that are required for endosomal Notch signaling ncode subunits of the V-ATPase, the molecular machine that creates a proton motive force to acidify most compartments of the endosomal system. The data thus establish a novel role of V-ATPase that is relevant to both physiological and pathological Notch signaling. While this work was under review, an article describing a similar role for V-ATPase regulators and a different subunit of the V-ATPase in Notch signaling was published (Yan, 2009). These independent results confirm those findings and expand them by reporting the ultrastructure of V-ATPase mutant cells, extending the role to tumor contexts that depend on excess Notch signaling activity and, importantly, providing evidence for the early endosome as the site of the requirement for V-ATPase activity. Interestingly, the aquaporin Big brain (Bib) was recently shown to promote Notch signaling at a similar step of Notch processing, and bib mutant clones, like V-ATPase mutant clones, show defects in endosomal acidification (Kanwar, 2008). It is possible that some other, as yet uncharacterized, V-ATPase function unrelated to its well-established role in proton pumping might be required for Notch signaling activation. Alternatively, the common acidification and Notch signaling phenotypes of V-ATPase and bib mutant cells might indicate that Notch signaling normally requires endosomal acidification (Vaccari, 2010).

The best-understood requirement for V-ATPase-dependent acidification is in promoting lysosomal degradation by ensuring the proper targeting, release and activation of lysosomal proteases. Indeed, this study found that Notch and other cargoes are trapped in a lysosome-like compartment in V-ATPase-deficient cells, where they accumulate rather than being degraded. The cargo-trapping phenotype resembles that seen in the presence of chemical inhibitors of lysosomal acidification, such as chloroquine, as well as in cells mutant for regulators of lysosomal genesis such as the HOPS components Dor and Car and the PIKFYVE Fab1. However, in striking contrast to these latter mutants, in which Notch signaling is largely unaffected, a substantial loss of Notch signaling is seen in V-ATPase-deficient cells. This indicates that the requirement for V-ATPase in Notch signaling must precede lysosomal entry (Vaccari, 2010 and references therein).

How could acidification of earlier endosomal compartments promote Notch signaling? One possibility is that the role of acidification reflects the requirement of endosomal transport for Notch activation. In mammalian cells, V-ATPases have been implicated in the recruitment of proteins that modulate traffic between early and late endosomes (Hurtado-Lorenzo, 2006). This study found a reduced rate of endocytosis in V-ATPase mutant cells, and it is possible that a dampened flux of Notch through the endocytic pathway contributes to reduced Notch signaling. However, it is believed that this role is unlikely to account for the V-ATPase mutant phenotype for the following reasons. First, although its endocytic rate may be reduced, Notch can clearly reach both early and late endosomal compartments in mutant cells; this mild quantitative reduction in traffic contrasts with the more substantial and qualitative loss of Notch signaling. Second, in mammalian cells, loss of endosomal acidification reduces progression between early and late endosomes. Since the requirement for Notch signaling is in entry into early endosomes, Notch activation would seem to be less sensitive to this step. Moreover, because Notch reaches late endosomal compartments in V-ATPase mutant cells, in Drosophila, at least, redundant mechanisms to energize endocytic traffic must exist. Third, the striking failure of Notch trapped within Hrs-positive early endosomes in V-ATPase ESCRT double mutants to signal suggests that even when endocytic traffic is blocked at the endosomal sorting step, Notch activation still requires V-ATPase activity. The absence of Notch activation in vps22 Vha55 cells is particularly striking because even when most Notch cannot reach endosomes in cells double mutant for the ESCRT component TSG101 (erupted) and the endosomal syntaxin avl (Syx7), the minor fraction of Notch that does reach endosomes is efficiently cleaved and strongly activates ectopic Notch signaling (Vaccari, 2008). This suggests that the role of V-ATPase in Notch activation goes well beyond trafficking. The data also reveal that it is not mere access to the early endosome that is required for Notch signaling, but rather that a specific physiological feature of that endosome is central to the signaling activation mechanism (Vaccari, 2010).

An attractive alternative is that V-ATPase-dependent acidification creates an endosomal environment that is conducive for productive Notch S3 cleavage and, therefore, signaling. In this scenario, Notch transits through the early endosome in V-ATPase mutant cells, but is not efficiently activated because of altered γ-secretase function. Such a role is consistent with genetic experiments, which show that a membrane-tethered Notch truncation that is automatically processed to an active form by γ-secretase in WT cells cannot signal in cells that lack V-ATPase function. Although it is appealing to speculate that an acidic pH could promote overall cleavage by γ-secretase, which requires low luminal pH for optimal activity, experiments testing the acid dependence of other γ-secretase substrates have failed to show a reduction in overall S3-type processing (Vingtdeux, 2007). Although these assays are limited in their ability to reflect the in vivo situation, an interesting possibility suggested by mammalian studies is that pH might alter the precision of the S3 cleavage site, producing NICD forms that are quickly degraded and cannot effectively signal (Tagami, 2008). Additional possibilities exist, such as that rather than creating a cleavage-promoting environment per se, the V-ATPase could serve as a pH sensor that couples the generation of a low pH to the recruitment of γ-secretase regulators in order to restrict cleavage to an endosomal site. Finally, acidification could directly affect the correct maturation and localization of the γ-secretase enzyme. Future work will distinguish between these possibilities (Vaccari, 2010).

Regardless of the specific mechanism governing the role of V-ATPase in Notch cleavage, overall the current data suggest that drugs that impair V-ATPase function and consequently reduce endosomal acidification might be used to curtail pathologic overactivation of Notch, such as that observed in cancers characterized by Notch overexpression (Roy, 2007). Considering that such a goal is presently being pursued by the use of γ-secretase inhibitors, the use of already established V-ATPase inhibitors, such as Bafilomycin A1, either alone or in combination with γ-secretase inhibitors, could represent a promising therapeutic avenue (Vaccari, 2010).

The vacuolar proton pump, V-ATPase, is required for notch signaling and endosomal trafficking in Drosophila

This study identifies Rabconnectin-3alpha and beta (Rbcn-3A and Rbcn-3B) as two regulators of Notch signaling in Drosophila. In addition to disrupting Notch signaling, mutations in Rbcn-3A and B cause defects in endocytic trafficking, where Notch and other membrane proteins accumulate in late endosomal compartments. Notch is transported to the surface of mutant cells, and signaling is disrupted after the S2 cleavage. Interestingly, the yeast homolog of Rbcn-3A, Rav1, regulates the V-ATPase proton pump responsible for acidifying intracellular organelles. Similarly, Rbcn-3A and B appear to regulate V-ATPase function. Moreover, mutants were identified in VhaAC39, a V-ATPase subunit, and it was shown that they phenocopy Rbcn-3A and Rbcn-3B mutants. These results demonstrate that Rbcn-3 affects Notch signaling and trafficking through regulating V-ATPase function, which implies that the acidification of an intracellular compartment in the receiving cells is crucial for signaling (Yan, 2009).

In a genetic screen in the follicular epithelium, mutations were identified in the fly homologs of the WD40 proteins Rbcn-3A and B, and a V-ATPase V0 d subunit, VhaAC39. It was shown that interfering with V-ATPase function leads to a block in Notch signaling (Yan, 2009).

Mammalian Rbcn-3A and B form a complex that interacts with both Rab3-GAP and Rab3-GEP, but the biological function of this complex has remained elusive. Interestingly, the closest homolog of Rbcn-3A in yeast is the protein Rav1, a component of the RAVE complex. The RAVE complex was shown to interact with the V1 subcomplex of the V-ATPase and promote its activity by regulating the assembly of the peripheral V1 and membrane V0 subcomplexes to form the V-ATPase holoenzyme. V-ATPases are evolutionarily conserved ATP-driven proton pumps responsible for the acidification of several intracellular compartments, including endosomes, lysosomes, secretory vesicles, and the Golgi apparatus. Inhibition of V-ATPase function leads to a failure in luminal acidification of these different compartments. Likewise, in Rav1 mutants in yeast, V-ATPase-dependent vacuolar acidification is disrupted. Similarly, Rbcn-3A and B mutant follicle cells in Drosophila fail to acidify intracellular compartments. A comparable lack of acidic compartments was seen in follicle cells mutant for the VhaAC39 gene, which encodes one of the two V-ATPase V0 d subunits. The V0 d subunit was suggested to regulate the coupling of ATP hydrolysis and proton translocation and is therefore indispensable for V-ATPase activity (Yan, 2009).

In eukaryotic cells, V-ATPase-dependent acidification of organelles is necessary for protein sorting, trafficking, and turnover. For instance, hydrolases responsible for protein degradation in the lysosome have an optimal activity at a low pH. In addition, several trafficking steps along the endocytic pathway have been shown to rely on V-ATPase function. Mutations in V-ATPase components or pharmacological inhibition of V-ATPase activity results in an accumulation of membrane proteins in endocytic compartments and in some cases blocks transport between the late endosome and the lysosome. Likewise, Rav1 mutants in yeast show an accumulation of endosomes and a delay in vacuolar transport and degradation. Consistent with these data, in Rbcn-3 mutant cells Notch and other integral membrane proteins accumulate in enlarged late endocytic compartments. An identical phenotype was seen upon disruption of VhaAC39 (Yan, 2009).

The RAVE complex was identified in yeast, but so far a similar complex has not been described in higher eukaryotes. By demonstrating a striking resemblance between Rbcn-3 and VhaAC39 mutant cells with respect to intracellular acidification and protein trafficking, evidence is provided for the existence of a similar complex in higher eukaryotes. This conclusion is supported by the observation that HA-tagged Rbcn-3B can be immunoprecipitated with at least two components of the V1 subcomplex, the B subunit Vha55 and the H subunit VhaSFD. In addition to Rav1, the yeast RAVE complex contains two other components: Rav2 and Skp1. Skp1 is a highly conserved SCF ubiquitin ligase that forms multiple distinct complexes involved in a wide array of cellular processes. Rav2 on the other hand has no obvious homologs in Drosophila or other higher eukaryotes. Conversely, no clear Rbcn-3B homolog exists in yeast. In rat, the Rav1 homolog Rbcn-3A forms a complex with Rbcn-3B, and based on the identical phenotypes of Rbcn-3A and B mutants in Drosophila they likely act in a complex in flies as well. An interesting possibility is therefore that Rbcn-3B performs the function of Rav2 in the Drosophila RAVE complex (Yan, 2009).

Interestingly, Rbcn-3 and VhaAC39 mutants were isolated in a screen because of their phenotypic similarity to mutations in Notch pathway components, and indeed it was confirmed that both Rbcn-3 and VhaAC39 are critical factors required for Notch signaling. During Drosophila oogenesis, Notch signaling is required for multiple processes. In particular, at stage 6 of oogenesis, Delta expressed in the germline signals to Notch in the follicle cells to initiate the switch from mitosis to endocycle. The loss of Rbcn-3 or VhaAC39 in follicle cells phenocopies defective Notch signaling with respect to the mitosis-endocycle switch. In addition, defects were observed in other Notch-dependent processes during oogenesis in the absence of Rbcn-3 or VhaAC39, including fused egg chambers and anterior-posterior polarity defects. It was found that Rbcn-3 also affects Notch signaling in eye discs. The results showing that disrupting either a V-ATPase subunit (VhaAC39) or a V-ATPase regulator (Rbcn-3) both lead to a loss of Notch signaling provide evidence for a role of the vacuolar proton pump in the regulation of Notch signaling (Yan, 2009).

How could the vacuolar proton pump regulate Notch signaling? Since the loss of Notch signaling is evident upon disruption of Rbcn-3 or VhaAC39 function in the follicle cells, the signal-receiving cells with respect to Notch signaling, it is clear that V-ATPase function must be required at the level of Notch or in a downstream signaling event. In the absence of Rbcn-3 or VhaAC39, Notch accumulates strongly in enlarged late endosomal compartments, consistent with the known role for V-ATPases in endocytic trafficking and lysosomal degradation. However, it is highly unlikely that the accumulation of Notch in this late endosomal compartment is responsible for the observed block in signaling. Defects in early endocytic trafficking of Notch have been correlated to aberrant Notch signaling. Mutations in proteins that affect the first steps in endocytosis, such as Clathrin heavy chain (Chc), the fly dynamin Shi, the Rab5 GTPase, and the syntaxin Avalanche lead to a loss of Notch activity. In these mutants, Notch accumulates at the cell surface and fails to reach the early endosome. In contrast, mutations in some proteins that affect sorting at the MVB, such as the endosomal sorting complex required for transport (ESCRT) components Tsg101 and Vps25, cause ectopic activation of the Notch pathway. In these mutants, Notch accumulates with ubiquitinated cargo in enlarged MVB. However, mutations in proteins acting at the late endosome, such as the Phosphatidylinositol 3-Phosphate 5-kinase Fab1, cause Notch to accumulate in the late endosome after deubiquitination has occurred. Significantly, whereas these late endosomal mutants show Notch accumulation in late endosomal compartments comparable to that seen in Rbcn-3 and VhaAC39 mutants, they do not perturb Notch signaling. This indicates that the accumulation of Notch in late endosomes upon disruption of V-ATPase activity cannot explain the loss of Notch signaling (Yan, 2009).

In addition to a role in endocytosis and lysosomal degradation, V-ATPases function in the secretory pathway, both at the level of protein sorting in the Golgi and in the fusion of secretory vesicles with the plasma membrane. It has also been observed that acidification of vesicles is important for recruiting certain cytosolic coat proteins. It was therefore possible that the loss of Notch signaling in Rbcn-3 or VhaAC39 mutant cells was caused by a defect in the trafficking of Notch to the cell surface. No evidence was found for a requirement of Rbcn-3 or VhaAC39 in exocytosis; Notch and other membrane proteins still reach the cell surface in the absence of Rbcn-3 or VhaAC39. Nevertheless, it remains possible that VhaAC39 or Rbcn-3 mutants show subtle defects in exocytic trafficking or in the posttranslational modification of Notch in the secretory pathway, which may then be responsible for a loss of signaling (Yan, 2009).

It is possible that the loss of Notch signaling in VhaAC39 and Rbcn-3 mutant cells is due to a defect in the trafficking and/or activity of another pathway component. A possible candidate is γ-secretase. γ-secretase-mediated S3 cleavage of NEXT, the Notch product generated upon ligand-induced cleavage by ADAM metalloproteases, results in the generation of the active form of Notch, NICD. It was shown that the expression of NICD, but neither full-length Notch nor NEXT, can rescue defective Notch signaling in Rbcn-3 mutant follicle cells. Furthermore, NEXT ectopically expressed in wing imaginal discs signals much less efficiently in the absence of Rbcn-3B. These results are consistent with a requirement for V-ATPase activity at the level of or downstream of the S3 cleavage. The γ-secretase complex consists of four core transmembrane proteins. The complex is assembled in the ER and shuttles between the ER and the Golgi. A small fraction of the γ-secretase complex is transported to the plasma membrane and endosomes where it is thought to mediate the cleavage of its substrates such as NEXT. It is conceivable that disrupting V-ATPase activity results in the aberrant trafficking of the γ-secretase complex, thus preventing the S3 cleavage of Notch (Yan, 2009).

Alternatively, it has been reported that γ-secretase activity is optimal at a low pH. Recent evidence also indicates that S3 cleavage of Notch can generate heterogeneous fragments that differ by a few amino acids with different stabilities, thus exhibiting different signaling potencies. Therefore, a loss of V-ATPase activity and the resulting alkalization of intracellular organelles could affect the generation or the release of the S3 cleavage product. In addition, a recent study suggested that mutations in the Aquaporin Big brain, which is required for Notch signaling, also show a reduced luminal acidification (Yan, 2009).

In summary, these results have demonstrated that regulating V-ATPase activity is fundamental to Notch signaling in Drosophila (Yan, 2009).

Mitf is a master regulator of the v-ATPase, forming a control module for cellular homeostasis with v-ATPase and TORC1

The v-ATPase is a fundamental eukaryotic enzyme that is central to cellular homeostasis. Although its impact on key metabolic regulators such as TORC1 is well documented, knowledge of mechanisms that regulate v-ATPase activity is limited. This study reports that the Drosophila transcription factor Mitf is a master regulator of the v-ATPase holoenzyme. Mitf directly controls transcription of all 15 v-ATPase components through M-box cis-sites and this coordinated regulation affects holoenzyme activity in vivo. In addition, through the v-ATPase, Mitf promotes the activity of TORC1, which in turn negatively regulates Mitf. Evidence is provided that Mitf, v-ATPase and TORC1 form a negative regulatory loop that maintains each of these important metabolic regulators in relative balance. Interestingly, direct regulation of v-ATPase genes by human MITF also occurs in cells of the melanocytic lineage, showing mechanistic conservation in the regulation of the v-ATPase by MITF family proteins in fly and mammals. Collectively, this evidence points to an ancient module comprising Mitf, v-ATPase and TORC1 that serves as a dynamic modulator of metabolism for cellular homeostasis (Zhang, 2015).

The vacuolar (H+)-ATPase (v-ATPase) is an evolutionary-conserved holoenzyme that controls basic cellular processes in eukaryotic cells. As an ATP-dependent proton pump, it acidifies intracellular or extracellular compartments and generates electrochemical gradients, with profound consequences on lysosomal degradation, the transport of metabolites across gut epithelia and many other cellular processes. In lysosomal metabolism, the v-ATPase is a dual player; its proton pumping ability establishes the low pH required by degradative enzymes, whereas its ATPase activity is essential for the aminoacid-dependent activation of TORC1 (the Target Of Rapamycin Complex 1 kinase that links lysosomal degradation to the nutritional state of the cell). Interestingly, in mammalian cell lines, both negative (Settembre, 2011) and positive (Pena-Llopis, 2011) correlation of TORC1 activity with v-ATPase gene expression (ATP6 genes) has been reported. Thus, the effect of TORC1 on the v-ATPase is unclear. However, in both cases, members of the MiT/MITF-family of transcription factors were implicated as mediators of positive or negative regulation by TORC1 (Zhang, 2015).

The four MiT-family genes of mammals, MITF, TFEB, TFE3 and TFEC, encode bHLH-Zip transcription factors that control basic cellular processes in eukaryotes as well as tissue identity and differentiation in animal development. Recent studies in mammalian cell lines have implicated MITF, TFEB and TFE3 in the regulation of degradation pathways. Expression profiling showed induction of lysosomal and autophagy genes by these factors, with most of the targets containing the CLEAR element, a binding sites for TFEB. Interestingly, the nuclear versus cytoplasmic localization of MITF, TFEB, and TFE3 is controlled by the TORC1 kinase through phosphorylation. The mTOR-associated Rag GTPases can interact at an N-terminal motif present in all three MiT factors and promote localization at the lysosome, where phosphorylation of the transcription factors by TORC1 then leads to their cytoplasmic sequestration by the 14-3-3 anchor protein . Alternatively, phosphorylation of TFEB by active TORC1 at a C-terminal serine-rich motif has been proposed to promote its nuclear translocation and activation. Different cell culture conditions and the complication of dealing with multiple MiT family members may have contributed to this discrepancy. Nonetheless, the nature of this TORC1 regulation needs further study (Zhang, 2015).

The invertebrate model organism D. melanogaster offers two advantages. First, it provides a sophisticated genetic model to address questions in vivo, and second, it has a single MiT-family factor. The gene Mitf (CG43369) is expressed broadly at a low level throughout the Drosophila life cycle, but is particularly enriched in the digestive system (Hallsson, 2004). Its physiological roles are unknown, due to a lack of loss-of-function analyses. This study identifies Mitf as a master regulator of the major cellular v-ATPase through transcriptional control of all 15 subunits of the holoenzyme. Modulation of gene expression is direct and impacts holoenzyme activity, with profound consequences on all three metabolic regulators. Mitf, the v-ATPase and TORC1 form a regulatory module that maintains the three factors in dynamic balance and may provide an adaptive feature to its regulation of metabolism. Interestingly, these Mitf functions appear to be conserved in human cells, pointing to an ancient MiT/v-ATPase/TORC1 module for cellular homeostasis (Zhang, 2015).

The role of the v-ATPase as a fundamental regulator of metabolism is well documented and is underscored by its requirement in all eukaryotic cells (Marshansky, 2014). Understanding its regulation and how this ties to major metabolic pathways is critical to decoding the complex mechanisms of cellular homeostasis. This study shows that Drosophila Mitf plays a major role in regulating v-ATPase activity. Regulation is at the transcriptional level and direct, through cis sites generally located just upstream of the promoter or in a large early intron of each subunit-encoding Vha locus. Strikingly, fifteen Vha genes appear to be organized into an Mitf-regulated synexpression group that ensures co-production of all components of the major vATPase. Through this mechanism, Mitf functions as a master regulator of the holoenzyme in the digestive system and other fly tissues (Zhang, 2015).

Concerted expression of v-ATPase loci has also been observed in vertebrates but the genetic and molecular mechanisms mediating this synexpression are largely unknown. Whereas the fly has a single Mitf gene, the situation in mammals is more complex due to the presence of TFEB and TFE3 as well. Nonetheless, in human melanoma cells and most likely in melanocytes, MITF appears to directly regulate a set of ATP6 genes for all main v-ATPase subunits. Fifteen ATP6 genes (encoding the 13 holoenzyme subunits and 1 accessory protein) are bound in both melanoma cells and primary melanocytes; among these, all show expression correlation with MITF in cell lines and most are downregulated in response to the partial silencing of MITF in cell culture. These 15 ATP6 genes are considered to be the most likely targets of direct regulation by MITF in melanoma and melanocytes (Zhang, 2015).

The remaining 10 loci show variable effects (with only one bound in melanocytes). These may include genes that are targets of other MiT factors in other tissues and can respond to MITF when it is overexpressed (as is often the case in melanoma tumors). In fact, it is likely that TFEB and TFE3 play a similar role as MITF in controlling most of the ATP6 loci in Hela cells and ARPE-19 cells, respectively. Further analyses of these TFE factors and ATP6 genes in these cell lines will show if this is the case (Zhang, 2015).

In Drosophila and other insects, the v-ATPase works at the plasma membrane of cells lining gut and Malpighian tubules to regulate pH, energize ion transport and modulate fluid secretion (Wieczorek, 2009). In the developing epithelia of eye, wing and in the ovary, pH modulates the activity of internalized receptors such as Notch; hence, v-ATPase activity has repercussions on signaling. In wing discs, mutations in VhaM8.9 can cause planar cell polarity defects, in addition to disrupting endosomal trafficking (Hermle, 2013). Whereas most of these functions would likely be affected in Mitf mutants,some v-ATPase subunits also fulfill specialized roles (Hiesinger, 2005). In the latter case, the influence of Mitf would depend on whether the specific subunit is under Mitf control and, if not, on whether the holoenzyme is the critical agent. Ultimately, many of these functions are essential for life and thus explain the lethality of Mitf mutant alleles (Zhang, 2015).

In mammals, the v-ATPase plays essential roles in a broad range of processes that are regulated by one or other MiT family member. The v-ATPase is essential for the proper function of melanosomes and many melanosome genes, in addition to ATP6 genes, are under MITF control. The v-ATPase also contributes to bone remodeling in osteoclasts, a cell type that expresses, and depends on MITF, TFEB and TFE3 for normal function. Interestingly, double knock-out of the Tfe3 and Mitf genes leads to osteopetrosis. The v-ATPase has also been found at the plasma membrane of cancer cells, from where it promotes alkalization of the cytoplasm and acidification of the tumour micro-environment, and this activity was recently linked to the emergence of distant metastases in melanoma. Further studies will elucidate the exact relationship between MiT factors and the v-ATpase in these contexts (Zhang, 2015).

Importantly, in both vertebrates and Drosophila, the v-ATPase mediates the activation of TORC1 at the lysosomal membrane in response to amino acids (Zoncu, 2011), thereby downregulating lysosomal metabolism. Hence, the v-ATPase can have a negative influence on the lysosome even though it promotes lysosomal function through acidification. In Drosophila, exogenous Mitf leads to increased TORC1 activity and promotes sequestration of Mitf back to the cytoplasm, whereas decreased v-ATPase gene dosage results in more nuclear Mitf as well as lower TORC1 function. In 501mel cell, exogenous MITF can also increase TORC1 activity. Although the predominant isoform of MITF in melanocytes does not have the Rag-binding sites, other isoforms do, as do also TFEB and TFE3. Hence, the regulatory loop is most likely conserved in mammals and functions in many cell types (Zhang, 2015).

Most importantly, Mitf does not merely execute a pro-lysosomal program when freed from TORC1-induced sequestration. Through the v-ATPase, Mitf feeds back onto TORC1 to promote and limit the activity of these important metabolic regulators. The Mitf/v-ATPase/TORC1 regulatory loop adjusts the activity of all three players offering a mechanism for continuously balancing metabolic pathways as the nutritional state of the cell fluctuates. In addition, it may confer an adaptive feature to the module. In this model, the level of v-ATPase, present at the lysosome, would sensitize or desensitize the nutritional sensing mechanism to changes in aa levels, thereby priming the system to reset at a new normal through TORC1 reactivation or inactivation. Such mechanism would impose a limit on upregulation of catabolism under lower nutrient conditions, and an upper limit on active TORC1 and its promotion of anabolic pathways when nutrients are abundant. Interestingly, cell culture experiments show that prolonged starvation reactivates TORC1; here, the loop offers a potential molecular mechanism for this effect. Evolutionarily, the Mitf/v-ATPase/TORC1 regulatory module may have conferred a selective advantage by fine-tuning the nutrient sensing mechanism to maintain metabolism within an optimal range in an ever-changing environment; an advantage particularly important for unicellular organisms or for cell types that require more precise metabolic regulation in multicellular ones (Zhang, 2015).

Drosophila provides an excellent metazoan model to investigate the molecular mechanisms for co-regulation of v-ATPase subunits as well as Mitf's contribution to the maintenance of cellular homeostasis. It will be also important to investigate how different members of the MiT family participate in these processes in different cell types under different physiological conditions and what impact they have on cellular homeostasis in health and disease (Zhang, 2015).

Nagata, R., Nakamura, M., Sanaki, Y. and Igaki, T. (2019). Cell competition is driven by autophagy. Dev Cell 51(1): 99-112. PubMed ID: 31543447

Cell competition is driven by autophagy

Cell competition is a quality control process that selectively eliminates unfit cells from the growing tissue via cell-cell interaction. Despite extensive mechanistic studies, the mechanism by which cell elimination is triggered has been elusive. Here, through a genetic screen in Drosophila, this study discovered that V-ATPase, an essential factor for autophagy, is required for triggering cell competition. Strikingly, autophagy is specifically elevated in prospective 'loser' cells nearby wild-type "winner" cells, and blocking autophagy in loser cells abolishes their elimination. Mechanistically, elevated autophagy upregulates a proapoptotic gene hid through NFkappaB, and the elevated hid cooperates with JNK signaling to effectively induce loser's death. Crucially, this mechanism generally applies to cell competition caused by differences in protein synthesis between cells. These findings establish a common mechanism of cell competition whereby cells with higher protein synthesis induce autophagy in their neighboring cells, leading to elimination of unfit cells (Nagata, 2019).

The data reveal a common mechanism of cell competition whereby cells with lower protein synthesis compared to their neighbors upregulate autophagy, which induces hid through NFκB, and the elevated hid cooperates with JNK to effectively cause cell death (see Molecular mechanism of cell competition triggered by relative differences in the protein synthesis). This phenomenon was commonly observed in both eye discs and wing discs using the ey-FLP and hs-FLP-mediated mitotic clone systems. Although it has been well documented that autophagy promotes cell survival, it also causes cell death in some cellular contexts. For instance, forced activation of autophagy in Drosophila imaginal discs or amnioserosa cells causes caspase-dependent cell death. Autophagy also drives programed cell death in salivary gland and midgut during Drosophila metamorphosis. Furthermore, similar to the current observations, JNK and autophagy were shown to cooperate to cause autophagic cell death in mammalian cells. Thus, a similar autophagy-mediated cell elimination could be involved in cell competition in mammals (Nagata, 2019).

The data show for the first time a cellular change, autophagy activation, is specifically induced in loser cells nearby winner cells as an early event in cell competition. The mechanism by which autophagy is specifically elevated at the clone boundary is currently unknown. A possible mechanism may be that loser cells with compromised protein synthesis actively uptake extracellular protein secreted by nearby wild-type winner cells, which could upregulate autophagy. Intriguingly, similar to Myc-overexpressing super-competitors, malignant RasV12/scribble-⁄- tumor cells upregulate autophagy in their surrounding wild-type cells non-autonomously, which is required for tumor growth and invasion. Thus, the current data suggest that autophagy-mediated cell competition may also drive tumor expansion during oncogenesis (Nagata, 2019).

It would be important to investigate the mechanism by which upregulated autophagy induces hid expression via NFκB in the future studies. Given that NFκB-mediated hid expression was shown to play a key role in executing Minute cell competition and Myc super-competition, the autophagy-mediated cell elimination machinery could also involve other regulators of Minute cell competition and Myc super-competition such as Fwe, Azot, and TRR signaling. Indeed, Hel25E-induced cell competition was significantly suppressed when Fwe, Azot, or a TRR signaling component was knocked down. On the other hand, suppression of Hel25E-induced cell competition by knockdown of Atg1, Azot, or Spätzle (Spz, a secreted ligand for TRRs), was not further enhanced by co-knockdown of two or three of them. These data suggest that autophagy, Azot, and TRR signaling components are converged to a single pathway. Further studies on the mdechanism by which these components cooperate to drive cell elimination would lead to a comprehensive understanding of cell competition (Nagata, 2019).

Genome-wide survey of V-ATPase genes in Drosophila reveals a conserved renal phenotype for lethal alleles

V-ATPases are ubiquitous, vital proton pumps that play a multiplicity of roles in higher organisms. In many epithelia, they are the major energizer of cotransport processes and have been implicated in functions as diverse as fluid secretion and longevity. The first animal knockout of a V-ATPase was identified in Drosophila, and its recessive lethality demonstrated the essential nature of V-ATPases. This article surveys the entire V-ATPase gene family in Drosophila, both experimentally and in silico. Adult expression patterns of most of the genes are shown experimentally for the first time, using in situ hybridization or reporter gene expression, and these results are reconciled with published expression and microarray data. For each subunit, the single gene identified previously by microarray, as upregulated and abundant in tubules, is shown to be similarly abundant in other epithelia in which V-ATPases are known to be important; there thus appears to be a single dominant 'plasma membrane' V-ATPase holoenzyme in Drosophila. This provides the most comprehensive view of V-ATPase expression yet in a multicellular organism. The transparent Malpighian tubule phenotype first identified in lethal alleles of Vha55, the gene encoding the B-subunit, is shown to be general to those plasma membrane V-ATPase subunits for which lethal alleles are available, and to be caused by failure to accumulate uric acid crystals. These results coincide with the expression view of the gene family, in which 13 of the genes are specialized for epithelial roles, whereas others have spatially or temporally restricted patterns of expression (Allan, 2005).

V-ATPases are large multisubunit pumps that transport hydrogen ions in exchange for energy, in the form of ATP. They are present in the endomembranes of all cells and in the plasma membranes of many specialized eukaryotic cells. The V-ATPases of animals, plants, and fungi are structurally very similar and are composed of two functional domains, V1 and Vo. The V1 domain, located on the cytoplasmic side of the membrane, is composed of eight different subunits (A-H) and is responsible for ATP hydrolysis. The Vo domain is a membrane-bound, proton-conducting complex composed of at least four, possibly five, subunits (a-e). In insects, V-ATPases are localized in the apical membranes of nearly all epithelial tissues (such as salivary glands, midgut, and Malpighian tubules), where they energize secondary active transport processes across the epithelium (Wieczorek, 2003). Tissue specificity has also been demonstrated for V-ATPase isoforms for the a- and c-subunits in Caenorhabditis elegans as well as the B-, C-, d-, E-, and G-subunits in mammals. This may reflect distinct targeting of isoforms in various tissues; indeed, differential localization of isoforms has been observed for the a-subunit in mice, where isoform-a1 resides in synaptic vesicles and -a3 in multinucleate osteoclasts (Allan, 2005).

The V-ATPase multigene family is encoded by 33 genes. Apart from five subunits in the V1 sector that are encoded by single genes (B, C, E, G, and H) and the accessory subunits vhaAC45 and vhaM8.9, each V-ATPase subunit is encoded by more than two genes, and as many as five genes encode the Vo subunits-a and -c. To add further complexity, seven subunits have been annotated by the genome project as encoding alternatively spliced genes, although splice variants have only been shown in the 5'-untranslated regions of the ESTs for four subunits. Therefore, splice variants that affect protein sequence may only occur for subunits-C, -H, and -a. Pseudogenes are thought to be rare in Drosophila, with perhaps only 100 in total, so it is likely that most of these genes will be authentic. EST corroboration of these transcripts is discussed below (Allan, 2005).

In some simple organisms, V-ATPase genes are found as an operon, allowing simple coordination of their expression. In Drosophila, however, the genes for most subunits are spread at apparently random chromosomal locations throughout the genome, although some subunits that are encoded by multiple genes show evidence of local gene duplication. For example, the A-subunit has a cluster of three genes at 34A, vha100-2 and vha100-4 are at 91A, vha16-2 and vha16-3 are at 68C, vhaPPA-1 and vhaPPA-2 are at 88D, and vhaM9.7-2 and vhaM9.7-3 are at 64B. Interestingly vha36-1 (CG8186) on the second chromosome has been described as a young retrotransposed gene derived from its parental gene vha36-3 (CG8310) on the X chromosome. The parental gene vha36-3 has three exons whereas the retrotransposed vha36-1 gene has one exon, presumably due to integration of reverse-transcribed mRNA into a new genomic position. Other V-ATPase subunits encoded by multiple genes have similar characteristics; the F-, c-, and e-subunits are encoded by multiple genes, of which one has multiple exons and the rest have one (Allan, 2005).

To map the expression of V-ATPase subunits, a 96-well in situ hybridization method was employed, using RNA probes directed against the 3'-UTR of each gene to minimize cross-hybridization. The results are informative at several levels. First, because the holoenzyme must be assembled with at least one gene product encoding each subunit, all genes that uniquely encode a subunit (e.g., B) are very widely expressed, as one would expect. Second, for each subunit, at least one gene showed strong hybridization signals in all major epithelial tissues (Malpighian tubules, midgut, hindgut, and rectum). In every case, these were the genes that had been implicated in Malpighian tubule function by virtue of tubule enrichment in a previous microarray analysis. Taken together, the microarray and in situ data thus allow the assertion that the plasma membrane V-ATPase holoenzyme, not just in Malpighian tubule but in the major transporting epithelia, is a single isozyme composed of polypeptides from vha68-2, vha55, vhaSFD, vha44, vha36-1, vha26, vha14-1, vha13, vha100-2, vha16-1, vhaPPA1-1, vhaM9.7-2, vhaAC39-1, and vhaAC45. This is the first time that a plasma membrane V-ATPase has been genetically characterized at this level (Allan, 2005).

Vha68-2 is among the V-ATPase genes that exhibit restricted tissue expression. Those genes considered to be plasma membrane V-ATPase subunits (vha68-2, vha55, vhaSFD, vha44, vha36-1, vha26, vhaM9.7-2, vhaAC39-1, and vhaAC45) generally show ubiquitous embryonic expression, with strongest staining in the gut and tubules. The other three genes (vha68-1, vha100-1, and vha100-4) show distinctly different patterns. In contrast to vha68-2, which shows expression in gut, tubules, caeca, and muscle, vha68-1 is restricted to hindgut, ventral nerve cord, and central nervous system. The fact that the majority of ESTs for vha68-1 are from head libraries (30/42), while head ESTs make up only one-third of vha68-2 ESTs (30/84) supports the assignment of vha68-1 as a head isoform. The gene vha68-3 has 56 ESTs that are exclusively derived from testes, and strong expression for this isoform was observed in the testicular ducts, accessory gland, ejaculatory duct, and bulb. The vha68 genes encoding the A-subunit thus show very clearly distinct roles for each gene (Allan, 2005).

In common with humans, the Drosophila a-subunit is encoded by multiple vha100 genes, and again there is some evidence for differential expression. The ubiquitous/epithelial isoform is encoded by vha100-2, while vha100-1 appears to be enriched in head, testes, and ovaries. Consistent with this, vha100-1 is most closely similar to the mammalian a1-isoform: in mouse, the a1-1-transcript is brain specific. All six vha100-3 ESTs obtained are from testes, suggesting tissue specificity for this isoform. The genes vha100-4 and vha100-5 are likely to have restricted embryonic expression: 12 of the 13 ESTs for vha100-5 are of embryonic origin, as well as the single EST for vha100-4. The expression pattern of vha100-4 observed from the online embryo expression database displays restricted expression in what appears to be copper cells of the gut. Thus the vha100 genes appear to be similar to worms, mice, Arabidopsis, and humans in that they have at least four isoforms with varying tissue expression. Because the a-subunit contributes to the hemichannels that couple rotation to proton transport by the holoenzyme, it is possible that selection of different a-subunits may provide different kinetic, or 'gearing,' properties appropriate to different tissues (Allan, 2005).

In situ results from this study, taken together with community in situ, lacZ, and EST data, show that the large majority of V-ATPase genes in Drosophila are genuinely expressed as mRNAs. For each subunit, there is a single gene that is widely expressed and that is also highly expressed in epithelia. Other genes appear to have developmentally or spatially much more restricted expression patterns, typically embryonic only, or specific for testes or head (Allan, 2005).

It has been previously shown that a known and well-characterized existing genetic locus (SzA) corresponds to the gene (vha55) encoding the B-subunit, thus making vha55 the first animal 'knockout' for a V-ATPase subunit (Davies, 1996). For this prototypic knockout, an allelic series was characterized. True nulls (deleted for the entire locus) survived to hatching but failed to thrive, whereas point mutant homozygotes could die midway through embryonic development. It has been suggested that these results are consistent with a large maternal investment of V-ATPase mRNA in the embryo, sufficient to permit hatching without further zygotic mRNA. By contrast, a wave of zygotic expression in midembryogenesis would be antimorphic if the mRNA encoded a defective protein. There is now gene expression evidence to support this dual-peak mRNA model. Using the BDGP gene expression website, it was observed that the plasma membrane V-ATPases have a large (necessarily maternal) mRNA expression from stages 1-5, followed by a sharp decrease in mRNA. Expression of zygotic V-ATPase mRNAs starts to rise again at stage 9. These results are exactly consistent with earlier predictions (Allan, 2005).

A characteristic of lethal alleles of vha55 is a transparent Malpighian tubule phenotype that is autonomous when mutant tubules are transplanted into abdomens of healthy flies. It has been suggested that this phenotype is due to a defect in urinary acidification. In insects, purine metabolites are excreted as uric acid, to conserve water. Urate ions are transported in soluble form into the tubule lumen, where they are acidified to precipitate uric acid. In normal embryos, birefringent uric acid crystals first become visible just before hatching (Allan, 2005).

If this explanation is correct, then the lethal clear tubule phenotype should be general to null alleles of all the plasma membrane V-ATPase subunits. Indeed, this phenotype has already been shown by null alleles of vha68-2 (Dow, 1997). The genetic resources available for Drosophila allow the generality of this hypothesis to be tested more comprehensively than has previously been possible for such a large gene family. P-element insertions were obtained for all the plasma membrane V-ATPase subunits except one (vhaAC39-1), as well as further EMS mutants for vha55, and both the lethality of the insertions and the lethal phases were established where appropriate, and a transparent tubule phenotype was scored. All the P-element insertions or EMS point mutations in V-ATPase genes except one (vha100-2) resulted in a lethal phenotype, mostly at the embryonic or early larval stage, and generally displayed clear tubule phenotypes. The only exceptions were P-element insertions in vha44, vhaPPA-1, and vhaAC45, which had much later lethal phases, suggesting that they were hypomorphs that expressed sufficient zygotic mRNA to survive hatching. This work thus shows that the transparent tubule phenotype is associated with disruption of any gene encoding a subunit of the tubule plasma membrane V-ATPase (Allan, 2005).

It is possible to confirm a previous model for the tubule phenotype. The tubule contains two kinds of apical concretion, spherites of calcium phosphate and crystals of uric acid; only the latter of these is birefringent under polarized light. The transparent tubule phenotype described is invariably associated with loss of birefringence in the tubule lumen; therefore, it results from failure to excrete uric acid (Allan, 2005).

The distribution of available lethal alleles among the V-ATPase genes is itself worthy of comment. Together with the available stocks, other workers have shown that P-element insertions in vha44 and vha100-2 are lethal. There are thus lethal alleles for all the subunits defined as plasma membrane, except vhaAC39-1, and there are no lethal alleles for any of the other genes. There are thus no mutants currently available to use as a negative control; so, while it was shown that lethal alleles of plasma membrane subunits do display the transparent tubule phenotype, the contrary cannot be asserted. Many tens of thousands of P-element insertion lines have now been generated across the world, and 40% of genes now have associated insertions. It would thus be tempting to suppose that the nonplasma membrane subunits do not have associated lethal alleles because their function is not essential. However, P-elements have notoriously nonuniform insertion patterns within the genome and prefer to insert in the first introns of abundantly expressed genes. Most of the nonplasma membrane subunits have only one exon and are thus difficult to disrupt by P-element insertions. It will be of interest to follow the progress of Drosophila gene disruption projects that utilize other classes of transposon, such as piggyBac (Allan, 2005).


Search PubMed for articles about Drosophila Vha68-2

Allan, A. K., Du, J., Davies, S. A. and Dow, J. A. (2005). Genome-wide survey of V-ATPase genes in Drosophila reveals a conserved renal phenotype for lethal alleles. Physiol Genomics. 22(2): 128-38. PubMed ID: 15855386

Davies, S. A., Kelly, D. C., Goodwin, S. F., Wang, S. Z., Kaiser, K. and Dow, J. A. T. (1996). Analysis and inactivation of vha55, the gene encoding the V-ATPase B-subunit in Drosophila melanogaster, reveals a larval lethal phenotype. J. Biol. Chem. 271: 30677-30684. PubMed ID: 8940044

Dow, J. A. T., et al. (1997). Molecular genetic analysis of V-ATPase function in Drosophila melanogaster. J. Exp. Biol. 200: 237-245. PubMed ID: 9050231

Hallsson, J. H., Haflidadöttir, B. S., Stivers, C., Odenwald, W., Arnheiter, H., Pignoni, F. and Steingrïmsson, E. (2004). The basic helix-loop-helix leucine zipper transcription factor Mitf is conserved in Drosophila and functions in eye development. Genetics 167: 233-241. PubMed ID: 15166150

Hermle, T., Guida, M. C., Beck, S., Helmstädter, S. and Simons, M. (2013). Drosophila ATP6AP2/VhaPRR functions both as a novel planar cell polarity core protein and a regulator of endosomal trafficking. EMBO J. 32: 245-259. PubMed ID: 23292348

Hiesinger, P. R., Fayyazuddin, A., Mehta, S. Q., Rosenmund, T., Schulze, K. L., Zhai, R. G., Verstreken, P., Cao, Y., Zhou, Y., Kunz, J. et al. (2005). The v-ATPase V0 subunit a1 is required for a late step in synaptic vesicle exocytosis in Drosophila. Cell 121: 607-620. PubMed ID: 15907473

Hurtado-Lorenzo A., et al. (2006). V-ATPase interacts with ARNO and Arf6 in early endosomes and regulates the protein degradative pathway. Nat. Cell Biol. 8: 124-136. PubMed ID: 16415858

Kanwar, R. and Fortini, M. (2008). The big brain aquaporin is required for endosome maturation and notch receptor trafficking. Cell 133: 852-863. PubMed ID: 18510929

Maegawa, Y., et al. (2006). Structure of the catalytic nucleotide-binding subunit A of A-type ATP synthase from Pyrococcus horikoshii reveals a novel domain related to the peripheral stalk. Acta Crystallogr. 62: 483-488. PubMed ID: 16627940

Marshansky, V., Rubinstein, J. L. and Grüber, G.(2014). Eukaryotic V-ATPase: novel structural findings and finctional insights. Biochim. Biophys. Acta. 1837: 857-879. PubMed ID: 24508215

Menut L., et al. (2007). A mosaic genetic screen for Drosophila neoplastic tumor suppressor genes based on defective pupation. Genetics 177: 1667-1677. PubMed ID: 17947427

Nagata, R., Nakamura, M., Sanaki, Y. and Igaki, T. (2019). Cell competition is driven by autophagy. Dev Cell 51(1): 99-112. PubMed ID: 31543447

Peña-Llopis, S., Vega-Rubin-de-Celis, S., Schwartz, J. C., Wolff, N. C., Tran, T. A. T., Zou, L., Xie, X.-J., Corey, D. R. and Brugarolas, J. (2011). Regulation of TFEB and V-ATPases by mTORC1. EMBO J. 30: 3242-3258. PubMed ID: 21804531

Roy, M., Pear W. S. and Aster J. C. (2007). The multifaceted role of Notch in cancer. Curr. Opin. Genet. Dev. 17: 52-59. PubMed ID: 17178457

Settembre, C., et al. (2011). TFEB links autophagy to lysosomal biogenesis. Science 332: 1429-1433. PubMed ID: 21617040

Tagami S., et al. (2008). Regulation of Notch signaling by dynamic changes in the precision of S3 cleavage of Notch-1. Mol. Cell. Biol. 28: 165-176. PubMed ID: 17967888

Vaccari, T., et al. (2008). Endosomal entry regulates Notch receptor activation in Drosophila melanogaster. J. Cell Biol. 180: 755-762. PubMed ID: 18299346

Vaccari, T., Duchi, S., Cortese, K., Tacchetti, C. and Bilder, D. (2010). The vacuolar ATPase is required for physiological as well as pathological activation of the Notch receptor. Development 137: 1825-1832. PubMed ID: 20460366

Vingtdeux V., et al. (2007). Intracellular pH regulates amyloid precursor protein intracellular domain accumulation. Neurobiol. Dis. 25: 686-696. PubMed ID: 17207630

Wieczorek, H., et al. (2003). The insect plasma membrane H+ V-ATPase: intra-, inter-, and supramolecular aspects. J. Bioenerg. Biomembr. 35: 359-366. PubMed ID: 14635781

Wieczorek, H., Beyenbach, K. W., Huss, M. and Vitavska, O. (2009). Vacuolar-type proton pumps in insect epithelia. J. Exp. Biol. 212: 1611-1619. PubMed ID: 19448071

Yan, Y., Denef, N. and Schüpbach, T. (2009). The vacuolar proton pump, V-ATPase, is required for notch signaling and endosomal trafficking in Drosophila. Dev. Cell 17(3): 387-402. PubMed ID: 19758563

Zhang, T., Zhou, Q., Ogmundsdottir, M. H., Moller, K., Siddaway, R., Larue, L., Hsing, M., Kong, S. W., Goding, C. R., Palsson, A., Steingrimsson, E. and Pignoni, F. (2015). Mitf is a master regulator of the v-ATPase, forming a control module for cellular homeostasis with v-ATPase and TORC1. J Cell Sci 128: 2938-2950. PubMed ID: 26092939

Zoncu, R., Bar-Peled, L., Efeyan, A., Wang, S., Sancak, Y. and Sabatini, D. M. (2011). mTORC1 senses lysosomal amino acids through an inside-out mechanism that requires the vacuolar H+-ATPase. Science 334: 678-683. PubMed ID: 22053050

Biological Overview

date revised: 30 August 2020

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