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We have identified Rabconnectin-3 alpha and beta (Rbcn-3A and B) as two regulators of Notch signaling in Drosophila. We found that, 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. We show that Notch is transported to the surface of mutant cells and that 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. We found that, similarly, Rbcn-3A and B appear to regulate V-ATPase function. Moreover, we identified mutants in VhaAC39, a V-ATPase subunit, and showed that they phenocopy Rbcn-3A and Rbcn-3B mutants. Our 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.
Notch signaling is a highly conserved cell communication pathway widely used in animal species (Artavanis-Tsakonas et al., 1999). Aberrant Notch signaling is associated with developmental disorders and cancers in humans (Lai, 2004; Talora et al., 2008). The core components of the Notch pathway are the single-pass transmembrane receptor Notch, its ligands and the Suppressor of Hairless transcription factor. Signaling is initiated upon ligand binding to Notch. This triggers two consecutive proteolytic cleavage events; a first extracellular cleavage mediated by ADAM-family metalloproteases, followed by an intramembranous cleavage by γ-secretase. As a result, the intracellular domain of Notch (NICD) is released and translocates into the nucleus to regulate transcription (Struhl et al., 1993; Rebay et al., 1993; Lai, 2004; Schweisguth, 2004).
The activity of both Notch and its ligands relies strongly on posttranslational modifications and intracellular trafficking (Le Borgne, 2006; Nichols et al., 2007; Stanley, 2007). Notch ligand activity requires the endocytic machinery. Similarly, Notch activation involves endocytic trafficking, but the precise mechanism by which endocytosis of Notch contributes to signaling activity remains unresolved. In Drosophila, mutations that block Notch trafficking at different endocytic steps have distinct effects on Notch signaling activity (Childress et al., 2006; Gallagher and Knoblich, 2006; Jaekel and Klein, 2006; Kanwar and Fortini, 2008; Moberg et al., 2005; Rusten et al., 2006; Seugnet et al., 1997; Thompson et al., 2005; Vaccari and Bilder, 2005; Vaccari et al., 2008). To explain the relation between Notch endocytic trafficking and Notch signaling activity, it has been proposed that Notch entry into early endosomes is critical for γ-secretase mediated Notch cleavage and thus normal Notch signaling, and that retention in the early endosome causes increased Notch signaling (Le Borgne, 2006; Nichols et al., 2007; Vaccari et al., 2008).
Here, we report the identification of the Drosophila homologues of mammalian Rabconnectin-3 alpha and beta (Rbcn-3A and B) and show that they are required for Notch signaling and endocytic trafficking in follicle cells and imaginal disc cells. In the absence of Rbcn-3A and B, Notch and other membrane proteins accumulate in an aberrant late endosomal compartment. Interestingly, the yeast homologue of Rbcn-3A, Rav1, was shown to regulate the assembly and activity of the vacuolar (H+) ATPase (V-ATPase) (Seol et al., 2001; Smardon et al., 2002). V-ATPases are ATP-driven proton pumps composed of two multi-subunit complexes: the membrane V0 complex and the peripheral V1 complex (Forgac, 2007; Jefferies et al., 2008). They are responsible for the acidification of intracellular compartments and have a well-established role in protein sorting, trafficking and turnover (Forgac, 2007; Jefferies et al., 2008). We show that the endocytic defects we observe in Rbcn-3A and B mutants are consistent with Rbcn-3 regulating V-ATPase activity. Moreover, we identify mutants in the V0 subunit VhaAC39 and show that their phenotypes with respect to Notch signaling and protein trafficking are identical to those in Rbcn-3 mutants. Our results indicate that Rbcn-3 acts primarily through regulating V-ATPase function and thus reveal a functional connection between the vacuolar proton pump and Notch signaling.
In a genetic mosaic screen aimed at identifying genes regulating the organization and morphogenesis of the Drosophila follicular epithelium (Denef et al., 2008), we isolated two complementation groups which caused defects reminiscent of those observed in follicle cells mutant for Notch. The two complementation groups were initially named after the representative alleles FK39 and FV10 and contained 13 and 17 alleles respectively.
Wild-type follicle cells divide mitotically from stage 2 to stage 6 of oogenesis and then switch from a mitotic cycle to an endocycle. From stage 7 to 10A follicle cells go through three rounds of DNA duplication without cell division (Horne-Badovinac and Bilder, 2005). This mitotic cycle to endocycle switch is regulated by the Notch pathway. Follicle cells in which Notch signaling is compromised keep dividing after stage 6 and fail to undergo endoreplication. As a result, follicle cell clones mutant for Notch form patches of cells with smaller nuclei than neighboring wild-type cells (Deng et al., 2001; Lopez-Schier and St Johnston, 2001). Follicle cell clones mutant for FK39 and FV10 show a similar phenotype (Figure 1A and data not shown). To confirm that the mutant cells continue dividing after stage 6 we stained for the mitotic marker phosphorylated Histone H3 (PH3) and found that, in contrast to wild-type egg chambers where PH3 positive cells are only found up to stage 6, PH3 staining was frequently present after stage 6 in these mutant cells (Figure 1B and data not shown).
The failure of FK39 and FV10 mutant follicle cells to switch from a mitotic cycle to an endocycle suggests defective Notch signaling. To test this more directly, we examined the expression of several known Notch targets in the follicular epithelium. The transcription factor Hindsight (Hnt) is upregulated by Notch signaling at stage 6/7 and this upregulation is required for the mitotic-cycle-to-endocycle switch in follicle cells (Sun and Deng, 2007). In contrast to wild-type cells Hnt upregulation was not detected in FK39 and FV10 mutant follicle cells in egg chambers beyond stage 6 (Figure 1C and data not shown). The homeodomain protein Cut is downregulated in wild-type follicle cells upon Notch activation at stage 6 and this downregulation is critical for follicle cells to enter the endocycle (Sun and Deng, 2005). In FK39 and FV10 mutant follicle cells Cut expression fails to be repressed after stage 6 (Figure 1D and data not shown). Finally, we examined the expression of a transcriptional reporter for Notch signaling, Gbe+Su(H)-lacZ (Furriols and Bray, 2001). In 77% of FK39 mutant clones (n=84), β-galactosidase staining was either absent or strongly reduced (Figure 1E). Taken together, these results indicate that Notch signaling is lost in FK39 and FV10 mutant follicle cells.
To ask whether the gene products disrupted in these mutants were required specifically in the follicular epithelium for Notch signaling or whether they also affected Notch signaling in other tissues, we examined the expression of the Gbe+Su(H)-lacZ reporter in FK39 mutant eye disc cells. Similar to our results in the follicle cells, we detected much lower levels of β-galactosidase in FK39 mutant eye disc cells compared to neighboring wild-type cells (Figure 1F, n>30), demonstrating that Notch signaling is compromised in FK39 mutant eye disc cells.
The observed loss of Notch signaling in FK39 and FV10 mutant follicle cells indicates that the gene products disrupted in FK39 and FV10 are required for Notch signaling in the signal receiving cells. Indeed, it was shown that the Notch ligand Delta is required in the germ line but not in the follicle cells to induce the mitotic to endocycle switch (Deng et al., 2001; Lopez-Schier and St Johnston, 2001). Upon ligand binding, Notch undergoes an extracellular cleavage (S2), generating the membrane bound NEXT, followed by an intramembranous cleavage (S3) producing NICD (Lai, 2004; Schweisguth, 2004). To further define the step where Notch signaling is disrupted in the mutants, we overexpressed full length Notch, NEXT or NICD constructs in FK39 mutant clones, using the MARCM system (Lee and Luo, 1999). With this system of clone generation, Hindsight failed to be upregulated in 83% of FK39 follicle cell clones (n=180) in stage 6/7 egg chambers, again indicating defective Notch signaling. Disrupted Notch signaling was similarly observed in 83% of FK39 clones with coexpression of full length Notch (n=121), and in 71% of FK39 clones which expressed NEXT (n=139), but only in 10% of FK39 clones which expressed NICD (n=122). (Figure 2A-D) Therefore, only the expression of NICD but not full length Notch or NEXT, can restore the loss of Notch signaling in FK39 mutant follicle cells.
In wing imaginal discs, overexpression of NEXT results in ectopic activation of the Notch target gene Cut (Rebay et al., 1993; Struhl and Greenwald, 1999). We therefore compared wing discs expressing NEXT in FK39 mutant clones with discs expressing NEXT in control clones. Cut was ectopically expressed in all cells of 59% of control clones (n=172) but only in 33% of FK39 mutant clones (n=152) (p<10-3, binomial probability) (Figure 2E, F). It is possible that perdurance of the wild-type gene product between the time of clone induction and dissection of the discs prevented a completely penetrant loss of signaling activity. Nevertheless, this result indicates that NEXT signals less efficiently in FK39 mutant cells than in control cells. We also attempted to assess whether ectopic Notch signaling induced by overexpession of NICD in wing discs could be suppressed by the loss of the FK39 gene product. However, we were unable to obtain a significant number of surviving FK39 clones expressing NICD in this experiment.
Taking together the evidence in both follicle cells and wing disc cells, we conclude that the gene product disrupted in FK39 mutant cells is required for signaling after the S2 cleavage of Notch but does not significantly affect Notch signaling after the release of the intracellular domain into the cytoplasm.
Using meiotic recombination with visible recessive markers and P element insertions we mapped the lethal phenotype of FK39 and FV10 to the chromosomal regions 2B15 and 5E respectively. The fact that these two complementation groups were mapped to distinct loci, yet shared identical phenotypes suggested that the two gene products might be functionally related. Two ORFs in these regions therefore stood out; CG3585 in region 5E and CG17766 in region 2B15 encode the fly homologues of the mammalian WD-40 proteins Rabconnectin-3 alpha and beta (Rbcn-3A and B) respectively. Rbcn-3A and B were identified as binding partners of Rab3-GEP and Rab3-GAP in synaptic vesicle extracts of rat brain (Kawabe et al., 2003; Nagano et al., 2002; Sakisaka and Takai, 2005). The two proteins appear to form a stable complex but the biochemical function of this complex remains unknown. The Drosophila Rbcn-3A gene was originally named DmX (Kraemer et al., 1998) and its mammalian homologues Dmx-like1 and Dmx-like2, with Rbcn-3A being Dmx-like2 (Kraemer et al., 2000; Nagano et al., 2002). Because of the functional connection between Rbcn-3A (DmX) and Rbcn-3B we will use the name Rbcn-3A in this paper.
To ask whether the loss of Rbcn-3A and B was indeed responsible for the phenotypes observed in FK39 and FV10 mutants, we sequenced the Rbcn-3B gene in seven alleles of the FK39 group and found that all of them contained mutations in Rbcn-3B (Figure 3A). Moreover, expression of a HA-tagged Rbcn-3B cDNA in FK39 mutant follicle cell clones rescued the Notch-like phenotype (Figures 3B and C, n>50). Similarly, a genomic rescue construct containing the Rbcn-3A gene and ~1.5 kb upstream and downstream sequences rescued the lethality and ovarian phenotypes of FV10 mutants (Figure 3D).
Taken together, our results demonstrate that the loss of Notch signaling observed in FV10 and FK39 mutants is caused by the loss of function of the Drosophila homologues of Rbcn-3A and Rbcn-3B respectively. Since the two genes share the same mutant phenotypes in all aspects of our analysis we will refer to them collectively as Rbcn-3, where appropriate.
We have shown that Rbcn-3 is required for Notch signaling in the receiving cells. Since effective Notch signaling relies on intracellular trafficking of Notch in the signal receiving cells (Le Borgne, 2006; Nichols et al., 2007) we asked whether Notch trafficking was defective in Rbcn-3 mutant cells. We therefore compared the subcellular distribution of Notch in Rbcn-3 mutant follicle cells and eye disc cells with that in wild-type cells. We found that in Rbcn-3 mutant follicle cells as well as eye disc cells, Notch accumulated in enlarged intracellular compartments (Figures 4A-E and data not shown). Notch accumulation in Rbcn-3 mutant cells was observed with antibodies against both the intracellular domain (NICD, Figures 4A, B and D) and the extracellular domain (NECD, Figures 4C and E), suggesting that full length Notch is present in these enlarged compartments.
We next asked whether the loss of Rbcn-3 specifically disrupts the distribution of Notch or whether other transmembrane proteins were also affected. We examined the subcellular localization of Delta (Figures 4F, G), EGF receptor (Supplemental Figures 1A, B), Domeless (Supplemental Figure 1C), Fas II (Figures 4H, I), FasIII and DE-Cadherin (data not shown) and found that they all accumulate in similar enlarged compartments in the absence of Rbcn-3. In contrast, membrane-associated proteins, such as aPKC, Crag and Dlg maintained a normal staining pattern in Rbcn-3 mutant follicle cells (Figures 4J, K and data not shown). Interestingly, despite the intracellular accumulation of the EGF receptor and of Domeless, which activates the JAK/STAT pathway, signaling downstream of these two receptors occurs normally in Rbcn-3 mutant follicle cells (Supplemental Figure 1).
The intracellular accumulation of Notch and other transmembrane proteins in Rbcn-3 mutant cells clearly demonstrates a role for Rbcn-3 in regulating protein trafficking. To ask whether the accumulation of membrane proteins in Rbcn-3 mutant cells is due to a defect in endocytic or exocytic trafficking, we performed a Notch trafficking assay in eye discs (Le Borgne and Schweisguth, 2003; Lu and Bilder, 2005). Rbcn-3B mosaic eye discs were incubated in cold medium containing an antibody against NECD to specifically label Notch at the cell surface. Pulse-labeled discs were either fixed immediately to examine the amount of Notch at the cell surface or incubated for different times in fresh medium at 25°C prior to fixation to follow endocytosis of labeled Notch. We did not observe a significant difference in the amount of Notch at the cell surface between wild-type and Rbcn-3B mutant cells, indicating that exocytosis of Notch is not noticeably affected in the absence of Rbcn-3B (Figure 5A and data not shown). Significantly, after a 1.5 h chase, abnormal accumulation of internalized Notch with bound antibody was observed in Rbcn-3B mutant cells compared to neighboring wild-type cells (Figure 5B). These results indicate that, in the absence of Rbcn-3, Notch reaches the surface of eye disc cells, is internalized, but then accumulates in an endocytic compartment.
A similar trafficking experiment is technically not possible in the follicle cells. To address whether Notch accumulation in Rbcn-3 mutant follicle cells results from defects in endocytic trafficking, as it does in eye disc cells, we compared Notch localization in Rbcn-3B mutant follicle cells with that in cells mutant for both Rbcn-3B and either Clathrin heavy chain (Chc) or shibire (shi), the fly orthologue of dynamin. Both Chc and Shi are required for the formation of endocytic vesicles at the plasma membrane (Ungewickell and Hinrichsen, 2007). In Chc and shi mutant follicle cells, Notch accumulates at the cell surface (Figure 5C, n>50 and data not shown)(Lu and Bilder, 2005; Vaccari et al., 2008). As described earlier, in Rbcn-3 mutant follicle cells Notch accumulates in enlarged intracellular compartments (e.g. Figures 4A-C, n>100). By contrast, in Rbcn-3B Chc and Rbcn-3B shi double mutant follicle cells Notch accumulated around the cell periphery as in Chc or shi single mutant follicle cells (Figure 5D, n>50 and data not shown). These results indicate that Rbcn-3 is not required for Notch to reach the cell surface and that the intracellular accumulation of Notch in the absence of Rbcn-3 requires Chc and Shi-dependent internalization.
Taken together, our results demonstrate that the major defect in Notch trafficking in Rbcn-3 mutant follicle cells and eye disc cells occurs in the endocytic pathway and that exocytosis occurs normally.
To determine the nature of the endocytic compartment in which Notch accumulates in the absence of Rbcn-3, we examined the distribution of known endocytic markers in Rbcn-3B mutant follicle cells and found that the enlarged compartments in mutant cells were positively labeled with the late endosomal marker YFP-Rab7 (Figure 5E) (Zhang et al, 2007), but not with the early endosomal markers GFP-Rab5 (data not shown) or GFP-2xFYVE (Figure 5F)(Wucherpfennig et al., 2003) or the recycling endosomal marker GFP-Rab11 (data not shown)(Emery et al., 2005). These results indicate that Notch and other membrane proteins accumulate in an enlarged late endosomal compartment in the absence of Rbcn-3.
To further characterize the nature of this late endosomal compartment, we stained egg chambers with an antibody detecting ubiquitinated proteins. Ubiquitination of membrane proteins often serves as a signal for their internalization and endocytic sorting prior to lysosomal degradation (Raiborg et al., 2003). In cells lacking components of the endosomal sorting complexes required for transport (ESCRTs), such as Hrs, Vps25 and Tsg101, which mediate protein sorting in the endocytic pathway, ubiquitinated proteins strongly accumulate in enlarged endosomes (Figure 5G) (Jekely and Rorth, 2003; Moberg et al., 2005; Thompson et al., 2005; Vaccari and Bilder, 2005). In contrast, the enlarged endosomes containing Notch and other membrane proteins in Rbcn-3B mutant cells do not contain increased levels of ubiquitin (Figure 5H).
Our data indicate that the loss of Rbcn-3 in Drosophila follicle cells causes membrane proteins to accumulate in enlarged late endosomes after deubiquitination has taken place. Intriguingly, other mutants that cause Notch accumulation in late endosomal compartments, such as the phosphatidylinositol-5-kinase Fab1, and the HOPS complex components carnation and deep orange, have no effect on Notch signaling (Rusten et al., 2006; Sevrioukov et al., 1999; Sriram et al., 2003 and data not shown). The accumulation of Notch in these enlarged late endosomal compartments in Rbcn-3 mutant cells can therefore not account for the observed loss of Notch signaling.
We have shown two major defects in Rbcn-3 mutant cells: a loss of Notch signaling and an accumulation of Notch and other membrane proteins in abnormal late endosomal compartments. Given that the late trafficking defect cannot explain the block in Notch signaling, we wanted to determine the underlying common cellular defect that could lead to these phenotypes. Upon closer inspection of the Rbcn-3A protein sequence we noticed that it shares homology with the yeast protein Rav1(Seol et al., 2001; Sipos et al., 2004), as well as the C. elegans Rav1 ortholog rbc-1 (wormbase). Rav1 is a component of the RAVE complex (regulator of the (H+)-ATPase of the vacuolar and endosomal membranes), which regulates the activity of the vacuolar (H+) ATPase (V-ATPase) (Seol et al., 2001; Smardon et al., 2002). The V-ATPases are conserved ATP-driven proton pumps composed of two multi-component subcomplexes, the membrane bound V0, which mediates proton translocation, and the peripheral V1, responsible for ATP hydrolysis (Forgac, 2007; Jefferies et al., 2008). The yeast RAVE complex promotes the assembly of V1 and V0 to form the active holo-enzyme (Seol et al., 2001; Smardon et al., 2002). One of the major functions of V-ATPases is to acidify the lumens of various intracellular organelles, including endosomes, lysosomes and secretory vesicles (Forgac, 2007; Jefferies et al., 2008). To test whether Rbcn-3A is required for this V-ATPase function in Drosophila as Rav1 is in yeast, we monitored the acidity of intracellular compartments with the pH-sensitive vital dye LysoTracker. In wild-type follicle cells acidic organelles were evident by intense LysoTracker staining (Figure 6A, B). By contrast, Rbcn-3A and Rbcn3-B mutant follicle cells showed strongly reduced or no LysoTracker staining (Figure 6A, B). This observation indicates a lack of acidic compartments in the absence of Rbcn-3 and is consistent with a role for Rbcn-3 in regulating V-ATPase-dependent acidification. In yeast, the RAVE complex was shown to bind the V1 subcomplex of the V-ATPase (Seol et al., 2001; Smardon et al., 2002). We therefore asked whether Rbcn-3 interacts with components of the V1 subcomplex in Drosophila. We immuno-precipitated two Drosophila V1 subunits, Vha55 (V1 B subunit) and VhaSFD (V1 H subunit) from ovaries expressing HA-tagged Rbcn-3B in follicle cells and found that HA-Rbcn-3B co-precipitated with both subunits (Figure 6C and data not shown). This result further confirms that Rbcn-3 could serve to regulate V-ATPase activity in Drosophila.
If Rbcn-3 indeed affects both Notch signaling and protein trafficking through regulating V-ATPase function, mutations in V-ATPase subunits should show similar defects as Rbcn-3 mutants. In the genetic screen that lead to the identification of Rbcn-3A and B, we found two additional mutants, FZ29 and FY38, that resulted in mosaic patches of cells with small nuclei. Upon closer inspection, we found that these mutants showed identical phenotypes to Rbcn-3 mutants. We mapped the lethality of these two mutants between two transposable elements at 3D4 and 4C2, and very close to the recessive marker echinus at 3F2 (Figure 7A). Interestingly, a single V-ATPase subunit gene, VhaAC39, maps exactly within this region (3F4) (Allan et al., 2005). We therefore sequenced the VhaAC39 gene in FZ29 and FY38, and found that, in both alleles, part of the VhaAC39 ORF was deleted. FY38 contains a 385 bp deletion combined with a 2 bp insertion and FZ29 contains a 137 bp deletion leading to a frame shift and a premature stop codon (Figure 7A and Supplemental Figure 2). VhaAC39 encodes one of the two Drosophila V0 d subunits (Allan et al., 2005; Nishi et al., 2003; Smith et al., 2002). As expected from the loss of VhaAC39 activity and thus V-ATPase function, LysoTracker staining was lost in VhaC39 mutant follicle cells (Figure 7B). In addition, Notch and other membrane proteins accumulated in enlarged intracellular compartments that were labeled strongly with the late endosomal marker GFP-Rab7 but not with the early endosomal markers GFP-Rab5, 2xFYVE-GFP, the recycling endosomal marker GFP-Rab11 or the Golgi marker Lava lamp (Figures 7C-F and data not shown). Very significantly, we found that Notch signaling was lost in the absence of VhaAC39; VhaAC39 mutant follicle cells keep dividing after stage 6, fail to upregulate Hnt, fail to downregulate Cut and show strongly reduced levels of Gbe+Su(H)-lacZ (Figure 8). Finally, VhaAC39 mutant eye discs cells behaved similarly to Rbcn-3 mutant eye disc cells in the Notch trafficking assay; Notch reached the surface, was internalized and accumulated in intracellular compartments (data not shown).
In summary, our results demonstrate that mutations in the V0 subunit VhaAC39 phenocopy mutations in Rbcn-3 with respect to protein trafficking and Notch signaling. We can therefore conclude that Rbcn-3 acts primarily through its effect on V-ATPase activity to regulate Notch signaling and endocytic trafficking. Moreover, our results provide evidence for a role of the vacuolar proton pump in the regulation of Notch signaling.
In a genetic screen in the follicular epithelium, we have identified mutations in the fly homologues of the WD40 proteins Rbcn-3A and B, and a V-ATPase V0 d subunit, VhaAC39, and have shown that interfering with V-ATPase function leads to a block in Notch signaling in Drosophila.
Mammalian Rbcn-3A and B were shown to form a complex that interacts with both Rab3-GAP and Rab3-GEF but the biological function of this complex had remained elusive (Kawabe et al., 2003; Nagano et al., 2002; Sakisaka and Takai, 2005). Interestingly, the closest homologue of Rbcn-3A in yeast is the protein Rav1, a component of the RAVE complex (Seol et al., 2001; Sipos et al., 2004). 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 (Seol et al., 2001; Smardon et al., 2002). 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 (Forgac, 2007; Jefferies et al., 2008). 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 (Seol et al., 2001). We have shown that, similarly, Rbcn-3A and B mutant follicle cells in Drosophila fail to acidify intracellular compartments. We showed a comparable lack of acidic compartments in follicle cells mutant for the VhaAC39 gene, which encodes one of the two V-ATPase V0 d subunits (Allan et al., 2005; Nishi et al., 2003; Smith et al., 2002). The V0 d subunit was suggested to regulate the coupling of ATP hydrolysis and proton translocation and is therefore indispensable for V-ATPase activity (Nishi et al., 2003; Smith et al., 2008).
In eukaryotic cells, V-ATPase dependent acidification of organelles is necessary for protein sorting, trafficking and turnover (Forgac, 2007; Jefferies et al., 2008; Nelson, 2003). 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 (Forgac, 2007; Marshansky and Futai, 2008). Mutations in V-ATPase components or pharmacological inhibition of V-ATPase activity result in an accumulation of membrane proteins in endocytic compartments and in some cases block transport between the late endosome and the lysosome (Nelson, 2003; van Deurs et al., 1996; van Weert et al., 1995). Likewise, Rav1 mutants in yeast show an accumulation of endosomes and a delay in vacuolar transport and degradation (Sipos et al., 2004). Consistent with these data, we have shown that in Rbcn-3 mutant cells Notch and other integral membrane proteins accumulate in enlarged late endocytic compartments. We observed an identical phenotype upon disruption of VhaAC39.
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 we have provided evidence for the existence of a similar complex in higher eukaryotes. This conclusion is supported by our observation that HA-tagged Rbcn-3B can be immuno-precipitated with at least two components of the V1 subcomplex, the B subunit Vha55 and the H subunit VhaSFD. In addition to Rav1the 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 homologues in Drosophila or other higher eukaryotes. Conversely, no clear Rbcn3-B homologue exists in yeast. In rat, the Rav1 homologue Rbcn-3A forms a complex with Rbcn3-B (Kawabe et al., 2003) 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.
Interestingly, we isolated Rbcn-3 and VhaAC39 mutants in our screen because of their phenotypic similarity to mutations in Notch pathway components and confirmed that indeed 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 (Deng et al., 2001; Lopez-Schier and St Johnston, 2001). We have shown that the loss of Rbcn-3 or VhaAC39 in follicle cells phenocopies defective Notch signaling with respect to the mitosis-endocycle switch. In addition, we observed defects in other Notch–dependent processes during oogenesis in the absence of Rbcn-3 or VhaAC39, including fused egg chambers and anterior-posterior polarity defects and found that Rbcn-3 also affects Notch signaling in eye discs. Our 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 provides evidence for a role of the vacuolar proton pump in the regulation of Notch signaling.
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. We have shown that, 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 Chc, the fly dynamin Shi, the Rab5 GTPase and the syntaxin Avalanche lead to a loss of Notch activity (Seugnet et al., 1997; Vaccari et al., 2008). In these mutants Notch accumulates at the cell surface and fails to reach the early endosome (Vaccari et al., 2008). 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 (Moberg et al., 2005; Thompson et al., 2005; Vaccari and Bilder, 2005; Vaccari et al., 2008). 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 (Rusten et al., 2006). 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 (Rusten et al., 2006; Vaccari et al., 2008). This indicates that the accumulation of Notch in late endosomes upon disruption of V-ATPAse activity cannot explain the loss of Notch signaling.
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 (Forgac, 2007; Marshansky and Futai, 2008). It has also been observed that acidification of vesicles is important for recruiting certain cytosolic coat proteins (Gu and Gruenberg, 2000). 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. We found no evidence for a requirement of Rbcn-3 or VhaAC39 in exocytosis and showed that 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.
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 (Lai, 2004; Schweisguth, 2004). We have 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, we have shown that 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 (Dries and Yu, 2008). 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 (Dries and Yu, 2008). 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.
Alternatively, it has been reported that γ-secretase activity is optimal at a low pH (Knops et al., 1995; Pasternak et al., 2004; Schrader-Fischer and Paganetti, 1996; Siman et al., 1993). Recent evidence also indicates that S3 cleavage of Notch can generate heterogenous fragments that differ by a few amino acids with different stabilities thus exhibiting different signaling potencies (Tagami et al, 2008). 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 (Campos-Ortega and Knust, 1990), also show a reduced luminal acidification (Kanwar and Fortini, 2008).
In summary, our results have demonstrated that regulating V-ATPase activity is fundamental to Notch signaling in Drosophila.
Rabconnectin-3A, Rabconnectin-3B and VhaAC39 mutations were isolated in a mosaic screen described by Denef et al. (Denef et al., 2008). In this screen, mutations were induced by EMS in y w FRT19 flies. The ChcGF23 and shiFL54 alleles used in the double mutant analysis were isolated in the same screen (N. D., Y. Y. and T. S., unpublished) and validated through complementation tests with the Chc1 and shiDN alleles (Bloomington). FRT40A hrsD28 flies were a gift from H. Bellen (Lloyd et al., 2002). Duplication and P-element lines used for mapping were obtained from Bloomington. Reporter lines used to assay signaling pathway activity are Gbe+Su(H)-lacZ (Furriols and Bray, 2001), mirror-lacZ (Xi et al., 2003) and kekkon-lacZ (Pai et al, 2000). The UAS-Notch line was a gift from G. Struhl. UAS-NEXT and UAS-NICD lines were gifts from S. Artavanis-Tsakonas (Rebay et al., 1993). Transgenic lines used to mark endocytic compartments are GFP-Rab5 (Wucherpfennig et al., 2003), GFP-Rab7 (Entchev et al., 2000), YFP-Rab7 (Zhang et al., 2007), GFP-Rab11 (Emery et al., 2005) and GFP-2xFYVE (Wucherpfennig et al., 2003). Follicle cell clones were generated using the FRT/UAS-Flp/GAL4 system (Duffy et al., 1998). Eye disc clones were generated using FRT/eyFlp. Follicle cell clones and wing disc clones which also express Notch constructs were generated using the MARCM system (Lee and Luo, 1999). The genotypes of flies and larvae used for the analyses are described in the Supplemental Data.
Ovaries and eye discs were dissected, fixed and stained by standard procedures. The Notch endocytic trafficking assay was conducted by following the procedure described in (Le Borgne and Schweisguth, 2003). For LysoTracker staining, ovaries were dissected, incubated in M3 culture medium containing 1μM LysoTracker Red DND-99 (Molecular Probes) for 5 min, rinsed three times with M3 medium and imaged immediately. Primary antibodies used are listed in the supplemental data. AlexaFluor 568 and 647 conjugated secondary antibodies and Hoechst were from Molecular Probes. Images were taken on a Zeiss LSM510 or a Leica SP5 confocal microscope.
Immuno-precipitation (IP) and western blotting was conducted by following the procedure described in (Pane et al., 2007). The antibodies used for IP were rabbit anti-vha55 (1: 500, (Du et al., 2006)) and anti-vhaSFD (1:500, (Pyza et al., 2004)). Both antibodies are kind gifts from J. A. Dow. The negative control was a non-specific rabbit pre-immune serum. The antibody used for western blotting was rat anti-HA-Peroxidase (1:3000, Roche).
Meiotic recombination mapping with visible recessive markers placed the lethal mutation in FV10 between crossveinless (5A13) and cut (7B4). It was further mapped between the P-elements BG02156 (5E1) and KG00403 (5E4). FK39 mutants were mapped using Duplication and Deficiency mapping. The lethality of FK39 mutants was rescued by Dp(1;Y)67g and complemented by the Deficiency lines Df(1)AD11, Df(1)S39, Df(1)6443, Df(1)Exel8196, Df(1)Pgd35 and Df(1)ED409 and by exclusion thus mapped to 2B15. PiggyBacs used for mapping of the lethal FZ29 and FY38 mutations were PBac(WH)CG16782f06392 (3D4) and PBac(WH)CG3626f02285 (4C2). To identify the molecular lesions in Rbcn-3B and VhaAC39 alleles, the mutant lines were balanced over an FM7, Kr>GFP balancer chromosome and genomic DNA from GFP- larvae (for Rbcn-3B) or embryos (for VhaAC39) was isolated. PCR products covering the Rbcn-3B or VhaAC39 gene regions of two independent genomic preps were sequenced and sequences were compared to FRT19 control sequences.
To make the Rbcn-3A genomic rescue construct, the Rbcn-3A gene and 1.5 kb upstream and downstream sequences were cloned from the BAC clone RP98-19D3 by recombineering cloning (Venken et al., 2006). To generate the pUASp-Rbcn-3B-HA construct, the coding region of Rbcn-3B (CG17766), which is contained in a single exon, was PCR amplified from the BAC clone RP98-5N9 and cloned into pUASp in frame with a C-terminal HA tag.
We thank H. Bellen, S. Bray, G. Struhl, S. Artavanis-Tsakonas, H. Krämer, S. Noselli, D. Harrison, M. Metztein, M. Gonzalez-Gaitan, JA. Dow, the Developmental Studies Hybridoma Bank and the Bloomington stock center for providing flies and antibodies; G. Barcelo for technique help; J. Goodhouse for advice with confocal microscopy and members of the Schüpbach and Wieschaus labs for feedback and advice. We also thank S. De Renzis and E. Wieschaus for helpful comments on the manuscript. This work was supported by the Howard Hughes Medical Institute and US Public Health Service Grant RO1 GM077620.
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