InteractiveFly: GeneBrief

Rabenosyn-5: Biological Overview | References

The small GTPase Rab5 has emerged as an important regulator of animal development and is essential for endocytic trafficking. However, the mechanisms that link Rab5 activation to cargo entry into early endosomes remain unclear. Rabenosyn (Rbsn) is shown to be a Rab5 effector that bridges an interaction between Rab5 and the Sec1/Munc18-family protein Vps45. The syntaxin Avalanche (Avl) was identified as a target for Vps45 activity. Rbsn and Vps45, like Avl and Rab5, are specifically localized to early endosomes and are required for endocytosis. Ultrastructural analysis of rbsn, Vps45, avl and Rab5 null mutant cells, which show identical defects, demonstrates that all four proteins are required for vesicle fusion to form early endosomes. These defects lead to loss of epithelial polarity in mutant tissues, which overproliferate to form neoplastic tumors. This work represents the first characterization of a Rab5 effector as a tumor suppressor, and provides in vivo evidence for a Rbsn-Vps45 complex on early endosomes that links Rab5 to the SNARE fusion machinery (Morrison, 2008).

The transport of protein cargoes to the numerous compartments within cells requires the budding, movement and fusion of membrane-bound vesicles. The myriad itineraries that vesicles follow require robust regulatory mechanisms to ensure specificity of delivery. One important site of regulation is at the fusion reaction itself. The core machinery that enables vesicle fusion consists of SNARE proteins, which are trans-membrane proteins located on the donor and target membranes that each contribute one of the four α-helices found in an assembled SNARE complex. Formation of a fusion-competent complex requires the incorporation of an α-helix from each of the different subfamilies of SNARE motifs, the Qa-, Qb-, Qc- and R-SNAREs (Fasshauer, 1998). Individual SNAREs within each subfamily are localized to distinct cellular compartments, suggesting that this distribution along with intrinsic SNARE pairing propensities may contribute to membrane fusion specificity. However, the properties of SNAREs alone appear insufficient to account for the specificity seen in vivo, indicating that other regulators are important to ensure the integrity of intracellular traffic (Morrison, 2008).

Rab proteins play a key regulatory role in SNARE-mediated fusion events. Like SNAREs, these small GTPases show distinct intracellular localization patterns and are required for specific transport steps (Stenmark, 2001). Rabs are thought to influence vesicle fusion by serving as molecular switches that, when activated, recruit additional factors -- Rab effectors -- to their site of action (Zerial, 2001; Grosshans, 2006). While activated Rabs generally bind to many different proteins, only a subset of these are actually direct effectors of vesicle trafficking. Identification of trafficking effectors requires a demonstration that the Rab and the effector are required for the same transport step. Genetic analyses in yeast have identified such proteins, in which loss-of-function phenotypes mimic those of mutations in specific Rabs and SNAREs. These trafficking effectors are structurally, and apparently functionally, diverse (Morrison, 2008).

Some effectors are thought to act as a physical 'tether' to mediate attachment between an incoming vesicle and its target membrane, bringing them into close proximity prior to vesicle fusion. Other effectors recruit proteins such as the Sec1/Munc-18 family (SM proteins), which bind and regulate the SNARE fusion complex itself; these modes may not be mutually exclusive. Since the mechanisms by which Rab activation controls membrane fusion are varied and unclear, a thorough understanding requires the identification of Rab trafficking effectors and the molecular interactions by which they link the Rabs to the SNARE complexes (Morrison, 2008).

Although yeast genetics has pioneered the determination of Rab effectors that mediate most stages of intracellular transport, an important exception is plasma membrane-to-early endosome traffic. This is a particularly significant step in metazoan organisms, where the internalization of cell surface proteins into the endosomal pathway regulates many critical cell-cell interactions, including signaling and adhesion. Current knowledge of the mechanisms underlying cargo delivery to early endosomes derives from several different approaches in mammalian cells, including biochemical interactions and in vitro reconstitution of endosomal fusion reactions, which have demonstrated the central role of Rab5 in this event. Intriguingly, these studies have also identified two effectors, EEA1 and Rabenosyn-5, which are recruited to endosomes by activated Rab5 and are associated, directly and indirectly respectively, with SNAREs (McBride, 1999; Nielsen, 2000). The indirect association of Rabenosyn-5 with SNAREs is through Vps45, an SM protein that binds various syntaxins in vitro (Nielsen, 2000; Full text of article). Despite these interactions, functional studies have not demonstrated that these proteins are required for plasma membrane-to-early endosome transport in vivo; the identity of the Rab5 effector that mediates this trafficking step thus remains unresolved (Morrison, 2008).

Drosophila has emerged as a valuable system to study endocytosis in vivo, in particular for the stage of early endosomal entry. Reverse genetics originally established that, as in mammalian cells, Drosophila Rab5 is required for this trafficking step (Wucherpfennig, 2003). Recently, a forward genetic screen identified mutations in a syntaxin, called Avalanche (avl), that cause a similar endocytic phenotype to that of Rab5 mutations (Lu, 2005). The endocytic defects of Rab5 and avl imaginal disc cells lead to a loss of epithelial architecture, and mutant tissues show dramatic overgrowth to form tumor-like cell masses; this phenotype is termed 'neoplastic'. To identify factors that link Rab5 activation to Avl-mediated vesicle fusion, a screen was performed for new mutations that produced the same tumorous phenotype (Menut, 2007). This study describes two previously uncharacterized genes, which encode the Drosophila proteins Rabenosyn (Rbsn) and Vps45; both are required for plasma membrane-to-early endosome trafficking. Genetics, ultrastructural analysis and biochemical interactions were used to link Rab5 and Avl activities through Rbsn and Vps45. These data are consistent with a model in which Rbsn, via Vps45 binding, functions as a Rab5 effector and tumor suppressor by mediating early endosomal fusion (Morrison, 2008).

The screen for mutations affecting epithelial polarity and proliferation identified many new complementation groups that control epithelial tissue architecture (Menut, 2007). This screen used the eyFLP/cell lethal system to generate eye imaginal discs composed predominantly of homozygous mutant cells in an otherwise heterozygous animal (hereafter 'mutant eye discs'). In this assay, eye discs mutant for the MENE(2L)-C complementation group consist of rounded and dramatically disorganized masses of cells. A similar phenotype is seen in the ovarian follicle cells. While wild-type follicle cells form a monolayered epithelium, MENE(2L)-C mutant cells multilayer and often invade the germ cell cluster. Staining for proteins normally localized to apical or lateral plasma membrane domains reveals that these domains are misspecified in MENE(2L)-C mutant cells. The normally apically restricted protein Atypical Protein Kinase C (aPKC) fails to remain distinct from Discs-Large-marked (Dlg) basolateral domains, indicating that apicobasal polarity is disrupted in these mutants. MENE(2L)-C mutant eye discs also show strong upregulation of Matrix Metalloprotease 1 (Mmp1) expression, which correlates with neoplastic transformation. Finally, larvae with MENE(2L)-C mutant eye discs do not pupariate but continue to feed during an extended L3 stage; during this time the eye discs grow to be significantly larger than wild-type eye discs. The polarity, proliferation, and gene expression phenotypes all resemble those seen in tissues mutant for previously characterized neoplastic tumor suppressor genes (nTSGs) including scribble (scrib) and Rab5. However, complementation tests showed that MENE(2L)-C was not allelic to any known tumor suppressor gene. Collectively, these phenotypes therefore indicate that MENE(2L)-C disrupts a novel Drosophila neoplastic tumor suppressor gene (Morrison, 2008).

MENE(2L)-C disrupts a protein related to human Rabenosyn-5 To identify the gene disrupted by MENE(2L)-C alleles, complementation tests were performed with chromosomal deficiency stocks and a small deficiency, Df(2L)Exel7034, was found that failed to complement the two extant MENE(2L)-C alleles. Sequencing of genes located within the genomic region deleted in Df(2L)Exel7034 revealed that each MENE(2L)-C allele carries a lesion in the gene CG8506, which encodes a 505 amino acid protein. MENE(2L)-C40-3 is a missense mutation altering the initiating ATG to ATA; the next in-frame ATG is located at amino acid 116. MENE(2L)-CX17 is a nonsense mutation that introduces a stop codon at amino acid 241. Both alleles show identical phenotypes in imaginal discs as well as in follicle cell epithelia, and animals either homozygous for each allele or hemizygous over Df(2L)Exel7034 die before the second larval instar. In addition, antibodies raised against a GST-CG8506 fusion protein recognize a polypeptide of the expected molecular mass of 56kD in wild-type larval extracts; this polypeptide is absent from extracts of MENE(2L)-C40-3 tissue. These results indicate that MENE(2L)-C40-3 and MENE(2L)-CX17 are null alleles of CG8506 (Morrison, 2008).

Sequence analysis revealed that CG8506 encodes a protein containing a number of conserved domains, including an N-terminal C2H2 zinc finger, a FYVE domain, two repeats of the tripeptide motif NPF, and several coiled-coil regions. BLAST searches indicate that CG8506 has significant homology to the human Rab5-binding protein Rabenosyn-5 (Nielsen, 2000), which contains each of these domains, although in a different arrangement. CG8506 is shorter than Rabenosyn-5 and lacks a C-terminal helical region, the NPF motifs are N-terminal in CG8506 while they are C-terminal in Rabenosyn-5, and CG8506 contains a single coiled-coil region. Nevertheless, these features are not found together in any other protein encoded by the fly genome. Therefore CG8506 is referred to as rabenosyn (Morrison, 2008).

Mammalian Rabenosyn-5 has been linked to both the endocytic and the recycling pathways in part by virtue of its ability to bind simultaneously to Rab5 and Rab4 (De Renzis, 2002; Naslavsky, 2004). To explore whether Drosophila Rbsn might be involved in these trafficking pathways, in vitro binding assays were performed using recombinant Rbsn and Rab GTPases. It was found that Rbsn binds to Rab5 specifically in its GTP-, but not GDP-bound form. By contrast, no significant binding base detected between Rbsn and Rab4 in either GTP or GDP-bound forms. Because Rab11 has been implicated in recycling pathways, tests were performed to see whether Rbsn could bind to Rab11, but no interaction was detected. It is concluded that Rbsn interacts specifically with the endocytic regulator Rab5 at early endosomes but not with Rab proteins that control recycling (Morrison, 2008).

It was also asked whether the results of in vitro binding experiments reflected the protein association in vivo, by examining the subcellular localization pattern of Rabenosyn relative to each of the Rab proteins. In cultured Drosophila S2 cells, Rabenosyn is found in discrete puncta which partially overlap with Avl-positive endocytic compartments. Expression of Rab5-YFP demonstrates Rbsn and Avl colocalization in Rab5-positive puncta, indicating that Rbsn localizes to early endosomes in response to Rab5 activation. By contrast, in cells expressing activated forms of Rab4 or Rab11, Rbsn and Avl colocalize in puncta that are mostly discrete from those marked by Rab4 or Rab11, indicating that Rbsn is not strongly recruited to recycling endosomes, consistent with the in vitro results (Morrison, 2008).

These above data suggest an association between Rbsn and the endocytic pathway, and disruption of several endocytic stages has been previously shown to perturb both cell polarity and cell proliferation control (Lu, 2005; Vaccari, 2005). Therefore directly whether Rbsn was required for endocytosis was directly tested. In wild-type imaginal disc cells, the apically localized transmembrane protein Notch is continuously endocytosed and lysosomally degraded; the endocytic transient population can be visualized as intracellular cytoplasmic puncta. However, in rbsn cells, Notch is present at greater than wild-type levels; a similar elevation is seen with the apical transmembrane protein Crumbs (Crb). To directly analyze cargo internalization, a trafficking assay was performed in living disc tissue. This assay pulse-labels cell surface Notch using an antibody against the Notch extracellular domain; endocytosis is then allowed to occur over varying chase periods. After 10 minutes of chase in WT cells, Notch is internalized and is found in early endosomes, while after 5 hours, no Notch signal remains. In contrast, in rbsn mutant cells no intracellular Notch puncta are seen after 10 minutes; instead Notch remains in the cell periphery and this localization persists even after 5 hours. This pattern strongly resembles that seen in rab5 mutants, but contrasts with that seen with the late-acting ESCRT mutants (Vaccari, 2005), in which Notch accumulates in enlarged endocytic compartments; it also contrasts with mutations in 'junctional scaffold' neoplastic tumor suppressor genes such as scrib, where no effect on Notch endocytosis is seen. The activity of a Notch reporter is reduced in rbsn mutant discs as in Rab5 and avl mutants; this is consistent with studies indicating that Notch that does not enter endosomes has reduced signaling function (Vaccari, 2008) and suggests that Notch accumulation is not involved in the rbsn tumor phenotype. Together, these results establish that rbsn is required for an early step in the endocytic pathway (Morrison, 2008).

The data indicate that rbsn has an endocytic mutant phenotype similar to Rab5 mutants, and Rbsn colocalizes with Rab5 at early endosomes and binds directly to Rab5-GTP. These results suggest that Rbsn might regulate Rab5-dependent fusion events at the early endosome. Interestingly, the Sec-1/Munc-18 (SM) protein Vps45 has been identified as a Rabenosyn-5 interacting protein (Nielsen, 2000). While the function of mammalian Vps45 is unknown, the yeast homolog Vps45p has a well documented requirement in biosynthetic Golgi-tolysosome traffic, and interacts with a Rabenosyn-5 like protein Vac1p (Cowles, 1994; Peterson, 1999). To test whether Rbsn might associate with a Vps45-like protein, a single clear Vps45 homolog was identified among the 4 SM proteins in Drosophila, that is encoded by the uncharacterized gene CG8228 (hereafter referred to as Vps45). An MBP-Vps45 fusion protein was expressed in bacteria, and it was found, using in vitro binding assays, that Vps45 strongly binds to Rbsn. These data suggest that Rab5 might regulate traffic through Rbsn-dependent Vps45 recruitment to the early endosome. The localization of Vps45 in animals has not been previously reported. To assess the in vivo localization of Vps45, an epitope-tagged Vps45 construct was expressed in S2 cells. In transfected cells, Vps45 shows a punctate pattern with only partial overlap to that of Rbsn and Avl. Interestingly, upon overexpression of Rab5-YFP, Vps45 relocalizes to the resultant enlarged endosomes; most Vps45 in these cells colocalizes with both Rbsn and Avl. Taken together with the in vitro binding data, this result suggests that Vps45 is recruited to the early endosome in response to Rab5 activation (Morrison, 2008).

Since yeast Vps45p is required for vacuolar but not endocytic traffic (Raymond, 1992; Bryant, 1998), attempts were made to determine whether the Rbsn-Vps45 interaction observed in Drosophila was relevant for function at the early endosome. No mutations in Vps45 have been reported in flies. However, by testing the uncharacterized neoplastic mutants isolated in the screen, it was found that MENE(3R)-B alleles fail to complement deficiencies that remove Vps45. Vps45 was sequenced and lesions were found in the coding region in each of the two MENE(3R)-B alleles. MENE(3R)-BJJ2 causes premature termination of the protein at amino acid 233 of the 574 amino acid coding region, and MENE(3R)-BGG11 converts valine 219 to a glutamine. Larvae homozygous for either allele die before the third instar, and the mutant eye imaginal disc phenotypes are indistinguishable, suggesting that both represent null alleles (Morrison, 2008).

The phenotypes of Vps45 mutant tissues were analyzed. As in rbsn mutants, staining for Notch and Crb showed that these protein levels were elevated in Vps45 mutant discs. To test whether Vps45 mutants might cause neoplastic transformation by blocking endocytosis in a manner similar to rbsn mutants, Notch trafficking was examined. Using live trafficking assays, it was found that Notch was not internalized in Vps45 mutant cells, and accumulated near the cell surface in a manner resembling that of both rbsn and Rab5 mutant cells. Moreover, the cell polarity, proliferation, Mmp1 expression and Notch signaling phenotypes were indistinguishable from those of rbsn mutant discs. These data suggest that in Drosophila, Vps45 function is indeed required for endocytic traffic and in particular for early endosomal stages (Morrison, 2008).

SM proteins are canonically thought to be trafficking regulators, able to bind to either SNARE proteins or complexes and govern fusion between vesicular and target membranes. In Golgi-to-vacuole traffic in yeast, the binding of Vps45p to Tlg2p is necessary for fusion into the vacuole (Nichols, 1998). Physical interactions between the Drosophila homologs of these proteins were tested and a strong interaction was confirmed between Vps45 and Syx16. However, only a small fraction of Drosophila Syx16 localizes to endosomes, and strong expression of a Syx16 RNAi transgene did not generate defects associated with disrupted endocytosis. Since similar expression of a Vps45 RNAi transgene shows strong endocytic defects resembling that seen in null mutant tissue, it was asked whether Vps45 might control endocytosis by associating with early endocytic SNAREs such as Avl. Weak but consistent binding was found between Vps45 and Avl as compared to Syx1 as a negative control; this binding was comparable to that seen with the annotated Drosophila homolog of Syx13, which interacts with Vps45 in humans and C. elegans (Morrison, 2008).

To evaluate whether Vps45 might regulate these SNAREs in vivo, genetic interaction studies were performed. Moderately reducing the protein levels of Vps45 using an RNAi construct expressed in the posterior compartment of the adult wing produces no obvious phenotype. However, removing one copy of the wild-type Vps45 gene to further reduce Vps45 protein levels results in an enhanced phenotype, including aberrant vein formation and ruffling of the posterior margin. Similar defects are also seen when Avl protein levels are reduced by RNAi, suggesting that this phenotype results from impaired endocytosis. The Vps45 RNAi sensitized background was used to test whether other genes might act in the Vps45- regulated endocytic pathway. It was found that removing one copy of Syx13 or Syx16 did not alter the Vps45 RNAi phenotype but removing one copy of avl, as well as Rab5 and to a lesser extent Rbsn, resulted in an enhancement similar to that produced by the removal of one copy of Vps45. Although weak, the interaction between Vps45 and rbsn was consistent; 64% of en>Vps45-IR; rbsn/+ wings show ectopic vein formation across the posterior cross vein, versus only 17% of en>Vps45-IR wings. Analogous results were seen when knocking down avl, further validating the interactions between these genes. Along with the strong phenotypic similarity of the mutants, these results suggest that Vps45 acts together with Rab5, Rbsn and Avl at the early endosome, and point to Avl as a regulatory target for Vps45 (Morrison, 2008).

The genetic and biochemical interactions described above suggest the hypothesis that Rab5, Rbsn, Vps45 and Avl act together to promote a single stage of endocytic traffic, involving fusion of incoming endocytic vesicles into the early endosome. If this hypothesis is correct, then cells lacking any of these proteins should block endocytosis at the same step and show similar disruption of endosomal structures. Since light microscopy does not allow the resolution required to clearly distinguish these structures, transmission and immunoelectron microscopy were used to test this hypothesis. Late-stage oocytes, which are large cells with a defined endocytic pathway required for uptake of yolk proteins, were tested. By making germ-line clones, oocytes mutant for Rab5, rbsn, Vps45, and avl were tested; all four mutants are defective in the formation of yolk granules. In WT oocytes, yolk granules are late endocytic structures that have a characteristic, electron-dense appearance resulting from condensation of internalized yolk proteins. These structures are strikingly absent from mutant oocytes. Because the lack of yolk granules could point to a prior block in the endocytic pathway, endocytic intermediates were analyzed by using an antibody against the yolk proteins, which are produced outside the oocyte, to trace a known endocytic cargo within oocyte vesicular compartments. In wild-type oocytes, yolk proteins are found in numerous endocytic compartments spanning a wide size range. In contrast, in Rab5, avl, rbsn and Vps45 mutant oocytes, yolk proteins are confined to small vesicles with a narrow size distribution; these are primarily found in dense accumulations in close proximity to the plasma membrane. The diameter of the vesicles, approximately 100 nm, is consistent with both the expected size of internalized clathrin-coated vesicles and the size of vesicles present in WT oocytes, and both electron-dense coated and uncoated vesicles are seen. Taken together, these data indicate that endocytic vesicles still form in the absence of Avl, Rab5, Vps45, or Rbsn; these vesicles can uncoat but nevertheless cannot fuse to form later endoyctic structures. The strong phenotypic similarity seen amongst all four mutants using immuno-electron microscopy support a model in which Vps45 and Rabenosyn act with Rab5 and Avl to promote vesicle fusion into the early endosome (Morrison, 2008).

This study has used forward genetics to identify and characterize two essential regulators of plasma membrane-toearly endosome traffic in Drosophila: Rbsn and Vps45. Rbsn and Vps45 are related to proteins implicated in endocytosis in mammalian cells, and their endocytic role in Drosophila has been definitively demonstrated in this study by direct analysis of cargo trafficking in null mutant tissue. Loss of either protein disrupts the flow of information from the activated small GTPase Rab5 to the trans-SNARE complex and blocks the fusion of endocytic vesicles into the endosome. The endocytic defect further causes mispolarization of epithelial cells and consequent overproliferation to form 'neoplastic tumors.' Although it has been shown that Rab5 acts as a Drosophila tumor suppressor (Lu, 2005), Rab5 has many effectors that regulate cellular processes as diverse as lipid metabolism, cytoskeletal organization, and cargo recycling. The demonstration that Rbsn and Vps45 are effectors of the tumor suppressive-activity of Drosophila Rab5 emphasizes that growth regulation requires endosomal fusion itself. These two proteins therefore extend the list of endocytic regulators that act as tumor suppressors, confirm the critical role of endocytosis in coordinating cell polarity and cell proliferation, and provide insight into the processes controlling entry into the early endosome (Morrison, 2008).

The mechanisms linking Rab-mediated vesicle targeting and SNARE-mediated vesicle fusion are among the least well understood events in cellular trafficking. Drosophila Rbsn is shown to be a Rab5 effector, binding to Rab5-GTP and localizing to early endosomes. Like Rab5, Rbsn is required for early endocytic entry, and rbsn and Rab5 mutants are phenotypically indistinguishable. In particular, this study used high resolution immuno-electron microscopy to identify the site of cargo trapping in cells completely lacking rbsn and Rab5 (as well as Vps45 and avl). These mutants show a striking accumulation of endocytic cargo-containing vesicles of a size consistent with plasma membrane-derived carrier vesicles; the absence of large endosomal compartments suggests that these vesicles fail to undergo fusion to form early endosomes (Rubino, 2000). Together, the genetic, biochemical and in vivo phenotypic data provide strong support for a model in which Rbsn is a Rab5 effector essential for endocytic vesicles to fuse into the early endosome. The severe endocytic block seen in rbsn tissue contrasts with the phenotype of mammalian cells depleted of the related human protein Rabenosyn-5 by RNA knockdown, which allow early endosomal entry but are defective in recycling cargo back to the plasma membrane (Naslavsky, 2004). The involvement of Rabenosyn-5 in the recycling pathway is supported not only by this phenotype, but also by its ability to bind Rab5 and the recycling regulator Rab4 simultaneously, prompting a model in which Rabenosyn-5 acts to coordinate cargo transfer from the early to recycling endosomes (De Renzis, 2002). Drosophila Rbsn, despite its strong association with Rab5-GTP, does not bind to Rab proteins known to regulate recycling, and while Rabenosyn-5 contains separate Rab4 and Rab5 binding domains (Eathiraj, 2005), these domains show homology to the same single domain in Drosophila Rbsn. It can be speculated that in mammalian Rabenosyn-5, duplication of the Rbsn Rab-binding domain followed by subsequent functional divergence led to its adoption into the recycling pathway, while the mammalian tethering protein and Rab5 effector EEA1 played a greater role in regulating early endosome entry (Christoforidis, 1999a). Although such an evolutionary scenario is possible, the possibility cannot be excluded that Rbsn plays a role in Drosophila recycling, particularly since the strong endocytic defects that were observed in rbsn mutants are upstream of, and thus prevent analysis of, the recycling pathway (Morrison, 2008).

This analysis of rbsn null mutant tissue demonstrates that Rbsn is required for vesicles to fuse into the early endosome. How does Rbsn promote vesicle fusion? The Drosophila SM protein Vps45, which binds to Rbsn, is required for the identical step of endocytosis as Rbsn and Rab5. Vps45 localizes to early endosomes, and this localized is increased by Rab5 overexpression. Recruitment of Vps45 by Rbsn bound to active Rab5 may create a high local concentration of Vps45 (Nielsen, 2000); once concentrated at the endosome, Vps45 could act on SNARE proteins to enable fusion of incoming carrier vesicles. In contrast to yeast, where Vps45p is required for lysosomal delivery of biosynthetic cargo (Cowles, 1994), this study shows that Drosophila Vps45 is required for trafficking and degradation of surface-derived cargo, thus identifying an SM protein that acts in the endocytic pathway (Morrison, 2008).

Gengyo-Ando has reported that C. elegans oocytes lacking homologs of Vps45 or Rbsn are defective in yolk uptake, but did not distinguish the precise stage of endocytic traffic blocked; moreover, they could not identify any syntaxin required for endocytosis and therefore could not determine a functional target of Vps45 (Gengyo-Ando, 2007. Full text of article). This study provides evidence that the endocytic syntaxin Avl is a key Vps45 target. A clear genetic interaction was found specifically between Vps45 and avl, as well as a weak physical interaction between Vps45 and both Avl and Syx13. While human and C. elegans Syx13 have been shown to bind Vps45 (Nielsen, 2000; Gengyo-Ando, 2007), orthologous relationships with Drosophila syntaxins are ambiguous: both Drosophila Avl and Syx13 are similar to human and C. elegans Syx13 as well as to human Syx7. The data demonstrate that Avl is required for the fusion event required for cargo entry into early endosomes; although RNAi experiments do not reveal a role for Drosophila Syx13 in endocytosis, further experiments will be needed to clarify the function of Syx13 in vesicle trafficking (Morrison, 2008).

The in vitro physical interactions observed between Vps45 and both Avl and Syx13 were notably weaker than that with Syx16. However, the data do not provide evidence for an endocytic role of Syx16. In addition, the significance of SM protein binding to an isolated SNARE remains unclear. While in most cases it correlates with SNARE complex assembly, in some instances this interaction is not necessary for function in vivo, and in others it is associated with inhibition of incorporation into a SNARE complex. Considering these scenarios, phenotypic analysis of mutant tissues completely lacking Vps45 demonstrates common phenotypes to those completely lacking Rab5, Avl, or Rbsn at the tissue, cellular and ultrastructural levels, indicating that Vps45 acts as a positive regulator of early endocytic SNAREs; this is also consistent with the enhancing nature of the genetic interactions. Moreover, these data argue that Avl is a component of the SNARE complex whose activity in vesicle fusion requires Vps45, establishing a functional link between Rab5 and SNAREs essential for early endosomal entry (Morrison, 2008).

Taken together, these genetic, phenotypic, and biochemical analyses provide strong support for a model in which Rbsn, by binding to Vps45 and Rab5, enables incoming cargo vesicles to fuse into the early endosome. This trafficking event is required for the proper control of surface levels of transmembrane proteins and has significant consequences for tissue development. Given that plasma membrane-to-early endosome trafficking is a process by which metazoan animals can control intercellular interactions, Rbsn may be an attractive target for cellular regulation of this event. Indeed, genetic interactions hint at a role for Rbsn in modulating several cell-cell interaction and communication pathways; future work will reveal whether Rbsn activity is modulated in specific contexts to achieve different developmental outcomes (Morrison, 2008).

Membrane protein location-dependent regulation by PI3K (III) and rabenosyn-5 in Drosophila wing cells

The class III phosphatidylinositol-3 kinase [PI3K (III)] regulates intracellular vesicular transport at multiple steps through the production of phosphatidylinositol-3-phosphate [PI(3)P]. While the localization of proteins at distinct membrane domains are likely regulated in different ways, the roles of PI3K (III) and its effectors have not been extensively investigated in a polarized cell during tissue development. This study, in vivo functions of PI3K (III) and its effector candidate Rabenosyn-5 (Rbsn-5) were examined in Drosophila wing primordial cells, which are polarized along the apical-basal axis. Knockdown of the PI3K (III) subunit Vps15 resulted in an accumulation of the apical junctional proteins DE-cadherin and Flamingo and also the basal membrane protein beta-integrin in intracellular vesicles. By contrast, knockdown of PI3K (III) increased lateral membrane-localized Fasciclin III (Fas III). Importantly, loss-of-function mutation of Rbsn-5 recapitulated the aberrant localization phenotypes of beta-integrin and Fas III, but not those of DE-cadherin and Flamingo. These results suggest that PI3K (III) differentially regulates localization of proteins at distinct membrane domains and that Rbsn-5 mediates only a part of the PI3K (III)-dependent processes (Abe, 2009).

Cell polarity along the apical-basal axis is essential for the function of epithelial cells. This polarity is formed and maintained by distinct localization of membrane spanning and associated proteins, to apical, lateral or basal membrane domains. Membrane proteins localized to the apical or basolateral plasma membrane are endocytosed into early and apical or basolateral endosomes. For example, horseradish peroxidase (HRP) administered to the apical cell surface is incorporated into the apical early endosome. By contrast, HRP or dimeric IgA administered to the basolateral cell surface or transferring receptor (TfR) in the basolateral domain are internalized into the basolateral early endosome, which remain distinct. Sorting of proteins for transcytosis, recycling and degradation takes place in these early endosomes. The proteins, incorporated into apical and basolateral early endosomes, meet in common endosomes, a process that can be observed within 15 min after the onset of internalization in MDCK cells. The significance of keeping the apical and basolateral early endosomes distinct is thought to ensure that proteins from the apical and basolateral plasma membrane remain apart before the sorting processes proceeds. Although it is plausible that the trafficking of proteins in distinct membrane domains is regulated differently, the factors involved in such a differential regulation remain elusive (Abe, 2009).

One of the key molecules regulating membrane trafficking is PI3K (III), a heterodimer of Vps34p and Vps15p/p150, which produces phosphatidylinositol-3-phosphate (PI(3)P). PI(3)P is found to localize with early endosome and internal vesicles of multivesicular bodies (MVBs) in mammalian cells in culture. Genetic and pharmacological analysis, using yeast and mammalian cells in culture, suggests that PI3K (III) is required for five distinct processes. These are: (1) the fusion of clathrin-coated vesicles and early endosomes as well as the fusion between early endosomes; (2) the recycling from early endosomes back to the Golgi complex or other destinations; (3) the entry of proteins into the lysosomal degradation pathway; (4) the formation of internal vesicles of MVBs and (5) autophagy. Moreover, inactivation of PI3K (III) by Vps34 mutation leads to an expansion of the outer nuclear membrane and an abnormal reduction of the LDL receptor at the apical membrane in C. elegans. In Drosophila, dVps34 mutation results in defective endocytosis of the apical membrane protein Notch and a defective onset of autophagy. It has been suggested that PI3K (III) utilizes different effectors at apical and basolateral endosomes. However, the role of PI3K (III) in the regulation of protein localization at different membrane domains has remained unclear (Abe, 2009 and references therein).

To understand the various functions of PI3K (III), it is crucial to clarify which downstream effectors are involved in each of the processes it regulates. PI3K (III) is thought to exert its function through the recruitment of proteins that contain PI(3)P-binding motifs such as FYVE or PX domains. Among such proteins, Rabenosyn-5 (Rbsn-5) has been shown to contribute to endosome fusion and recycling processes in mammalian cells. Genetic studies on C. elegans and Drosophila also show that Rbsn-5 is essential for receptor-mediated endocytosis and endosome fusion, although it is not clear whether or not Rbsn-5 is involved in other PI3K (III)-related phenomena (Abe, 2009).

To determine how the proteins in distinct membrane domains are regulated by PI3K (III) and its effector Rbsn-5 this study analyzed Drosophila wing development. This provides a good model since wing primordial cells have a clear polarity along the apical-basal axis. In addition a number of membrane proteins are known to be transported in an organized manner along the apical-basal axis. For example DE-cadherin, a cell adhesion protein and Fmi, a planar cell polarity (PCP) core protein, are localized in the apical junctions or zonula adherens (ZA), whereas the cell adhesion molecules FasIII and β-integrin are localized in lateral and basal membranes, respectively. This study found that inactivation of PI3K (III) in the wing primordial cells by knockdown of dVps15 affects the localization of these membrane proteins differently. In particular, it was found that dVps15 knockdown results in the accumulation of FasIII at the lateral membrane, whereas it results in intracellular accumulation of DE-cadherin, Fmi and β-integrin. Importantly, inactivation of Rbsn-5 shows accumulation of FasIII and β-integrin at the lateral membrane and intracellular vesicles, respectively, but no effects of DE-cadherin and Fmi localization (see in contrast Mottola, 2010). These results provide evidence for a differential regulation of protein localization by PI3K (III) and Rbsn-5 at distinct membrane domains (Abe, 2009).

This study demonstrated that PI3K (III) differentially regulates the localization of proteins at distinct membrane domains. The intracellular accumulation of Fmi, DE-cadherin and β-integrin induced by the dVps15 knockdown might be due to defects in the degradation pathway, since the maturation of MVBs and the lysosomal trafficking were defective in these cells. However, unlike these proteins, Fas III did not accumulate in the intracellular compartments, but rather accumulated at the surface of the lateral plasma membrane. It is possible that PI3K (III) regulates proteins at the lateral membrane differently from those localized at other membrane domains. It is also possible that PI3K (III) regulates Fas III in a different way, irrespective of the membrane domain to which it is localized. Whichever is the case it will be important to elucidate the mechanism underlying this difference in a future study (Abe, 2009).

Rbsn-5, a FYVE domain-containing protein, shares a part of the functions of PI3K (III), in that it is necessary for the regulation of Fas III and β-integrin localization, but not that of DE-cadherin and Fmi localization. Although the Rbsn-5^C241 null mutant clones may not completely lack Rbsn-5 activity, the requirement of Rbsn-5, or at least the requirement of an appropriate amount, differs between these proteins with respect to normal trafficking. It appears that Rbsn-5 preferentially controls the events at the basolateral regions, given that Rbsn-5 is necessary for the formation of large endosomes at the basal region, whereas it is indispensable for the formation of actin bundles at the apical surface (Abe, 2009).

PI3K (III) has been implicated in the differential regulation of vesicle trafficking at apical and basolateral regions. For instance, a reduction of PI(3)P dissociates EEA1, a FYVE-domain containing protein essential for early endosome fusion, selectively from basolateral endosomes. However, which proteins, including EEA1, regulate the different trafficking pathways downstream of PI3K (III) has remained unknown. Rbsn-5 has been proposed to be a PI3K (III) effector, since Rbsn-5 harbors a FYVE domain. The current results provide further evidence supporting a possible functional interaction between these two molecules, based on their genetic interaction on the wing morphogenesis and the PI3K (III)-dependent Rbsn-5 immunostaining. Importantly, the different requirement of Rbsn-5 for trafficking at apical junction and basolateral membrane domains suggests that Rbsn-5 may a selective regulator under the control of PI3K (III) (Abe, 2009).

A novel function for the Rab5 effector Rabenosyn-5 in planar cell polarity

In addition to apicobasal polarization, some epithelia also display polarity within the plane of the epithelium. To what extent polarized endocytosis plays a role in the establishment and maintenance of planar cell polarity (PCP) is at present unclear. This study investigated the role of Rabenosyn-5 (Rbsn-5), an evolutionarily conserved effector of the small GTPase Rab5, in the development of Drosophila wing epithelium. It was found that Rbsn-5 regulates endocytosis at the apical side of the wing epithelium and, surprisingly, a novel function was discovered of this protein in PCP. At early stages of pupal wing development, the PCP protein Flamingo (Fmi) redistributes between the cortex and Rab5- and Rbsn-5-positive early endosomes. During planar polarization, Rbsn-5 is recruited at the apical cell boundaries and redistributes along the proximodistal axis in an Fmi-dependent manner. At pre-hair formation, Rbsn-5 accumulates at the bottom of emerging hairs. Loss of Rbsn-5 causes intracellular accumulation of Fmi and typical PCP alterations such as defects in cell packing, in the polarized distribution of PCP proteins, and in hair orientation and formation. These results suggest that establishment of planar polarity requires the activity of Rbsn-5 in regulating both the endocytic trafficking of Fmi at the apical cell boundaries and hair morphology (Mottola, 2010).

This study uncovered a novel role of Rbsn-5 in the establishment of PCP during pupal wing development, and it was further demonstrated that the PCP protein Fmi undergoes endocytic trafficking in a process that is dependent on Rbsn-5 and required for the establishment of PCP (Mottola, 2010).

Rbsn-5 shares with its mammalian orthologue Rabenosyn-5 several structural features, as well as the function of molecular coordinator of endocytosis and recycling. First, the inhibition of fluid-phase endocytosis observed in rbsn³⁴ cells is consistent with the impairment of early endocytic transport described for both Rabenosyn-5 and Vps45 in mammalian cells, C. elegans and Drosophila. Second, although Rbsn-5 does not bind Rab4, it interacts with EHD/RME1, a protein that is required for recycling cargo from endosomes to the surface. Third, the formation of expanded Rab5-positive endosomes in Rbsn-5 mutant cells phenocopies the endosomal enlargement observed upon inhibition of recycling. Moreover, the accumulation of Fmi in late endocytic compartments, which is also consistent with the requirement of Rbsn-5 (and the yeast orthologue Vac1p) for protein sorting to the degradative pathway, resembles the phenotype previously described for sec5^E13 clones in Drosophila oocytes (Mottola, 2010).

The function of Rbsn-5 in endocytic transport is required for the re-distribution of Fmi between endosomes and the apical cell boundaries during the establishment of PCP in the Drosophila wing. Before PD asymmetry is established in the whole tissue, endogenous Fmi is detected on Rbsn-5- and Rab5-positive early endosomes. At later stages, Fmi must recycle back to the plasma membrane because it subsequently localizes to the apical cell boundaries concomitantly with Rbsn-5. Recycling from endosomes to the cell surface is also consistent with the dependence on Fmi for the recruitment of the exocyst subunit Sec5 at the apical cell boundaries. Loss of Rbsn-5 causes intracellular accumulation of Fmi, which correlates with defects in PD polarity and hair orientation. Consistently, Rab5 overexpression, which influences Rbsn-5 redistribution, also alters Fmi trafficking and causes PCP defects. These data therefore indicate that Rbsn-5-dependent trafficking of Fmi is relevant for the establishment of PCP. Clearly, the data do not exclude the possibility that other (e.g. biosynthetic) trafficking events of Fmi might contribute to this process (Mottola, 2010).

Why is Fmi endocytosed and recycled during establishment of PCP in the pupal wing? It has been recently proposed that a combination of polarized secretion, Fmi endocytosis and stabilization of Fmi and Fz to the distal apical cell boundaries might underlie the establishment of cellular asymmetry. In line with this proposal, Rbsn-5-dependent trafficking might be required to remove unstable Fmi (associating only to Fz) from the apical cell boundaries and relocate it in regions of the plasma membrane where it can be stabilized in proximal PCP complexes. Therefore, the weak proximal non-autonomy in trichome orientation observed for rbsn³⁴ clones, which resembles the phenotype of fmi clones rescued with a GFP-tagged Fmi mutant lacking the cytoplasmic domain (Fmiδintra-EGFP), might be explained with the blockade of Fmi at the plasma membrane preferentially bound to Fz:Dsh complexes (Mottola, 2010).

Some defects observed for rbsn³⁴ clones, such as Fmi redistribution as swirls and proximal perturbation of trichome polarity inside mutant clones, together with weak proximal non-autonomy are also reminiscent of defects in Fat and Ds mutant clones. Interestingly, it was observed that big rbsn³⁴ clones could be found only on the distal side of the pupal wing. This might reflect a less important requirement for Rbsn-5 on this side compared with the proximal one. However, whether Rbsn-5 is also involved in the global propagation of PCP signalling via the upstream module Ds-Fat-Fj remains to be determined (Mottola, 2010).

Additionally, Rbsn-5 mutant clones show defects in hair formation and elongation. As endocytosis and actin cytoskeleton remodelling are functionally connected, these defects might be indirect consequences of endocytosis impairment. However, the specific accumulation of Rbsn-5 at the bottom of emerging hairs would be consistent with the idea that Rbsn-5 mediated endocytic/recycling trafficking might actively contribute to outgrowth of wing hairs, possibly by regulating specific membrane delivery (Mottola, 2010).

While preparing this manuscript, a study on the regulation of membrane protein localization by PI3K (III) and Rabenosyn-5 in Drosophila wing cells reported (Abe, 2009) that loss-of-function mutation of Rbsn-5 does not affect Fmi localization and hair formation and orientation. The discrepancy with the current data could be explained by the fact that, in that study, the analysis was conducted at 25°C instead of 18°C. Indeed, it was noticed that rbsn³⁴ clones are less healthy and tend to be smaller when grown at higher temperatures. Under these conditions, the intracellular accumulation of Fmi might well be less noticeable and the rate of lysosomal degradation may be higher (Mottola, 2010).

In conclusion, the characterization of Rbsn-5 during Drosophila wing development allowed discovery of a novel function for this Rab5 effector in vivo in a developmental context and provided evidence in favor of a role of the apical endocytic trafficking of Fmi in the establishment of PCP. Future studies will hopefully provide additional molecular links and mechanistic insights into the functional interplay between the endocytic and the PCP machineries (Mottola, 2010).

The endocytic pathway acts downstream of Oskar in Drosophila germ plasm assembly

Cell fate is often determined by the intracellular localization of RNAs and proteins. In Drosophila oocytes, oskar (osk) RNA localization and the subsequent Osk synthesis at the posterior pole direct the assembly of the pole plasm, where factors for the germline and abdomen formation accumulate. osk RNA produces two isoforms, long and short Osk, which have distinct functions in pole plasm assembly. Short Osk recruits downstream components of the pole plasm, whose anchoring to the posterior cortex requires long Osk. The anchoring of pole plasm components also requires actin cytoskeleton, and Osk promotes long F-actin projections in the oocyte posterior cytoplasm. However, the mechanism by which Osk mediates F-actin reorganization remains elusive. Furthermore, although long Osk is known to associate with endosomes under immuno-electron microscopy, it was not known whether this association is functionally significant. This study shows that Rabenosyn-5 (Rbsn-5), a Rab5 effector protein required for the early endocytic pathway, is crucial for pole plasm assembly. rbsn-5^- oocytes fail to maintain microtubule polarity, which secondarily disrupts osk RNA localization. Nevertheless, anteriorly misexpressed Osk, particularly long Osk, recruits endosomal proteins, including Rbsn-5, and stimulates endocytosis. In oocytes lacking rbsn-5, the ectopic Osk induces aberrant F-actin aggregates, which diffuse into the cytoplasm along with pole plasm components. It is proposed that Osk stimulates endosomal cycling, which in turn promotes F-actin reorganization to anchor the pole plasm components to the oocyte cortex (Tanaka, 2008).

The polarized targeting and anchoring of specific molecules and organelles to particular subcellular regions are crucial for many cellular processes, including cell-polarity establishment and cell-fate determination. In many animals, germline fate is controlled by maternal factors localized to a specialized cytoplasmic region within the egg, called the germ plasm. Germ plasm contains germ granules, which are electron-dense, and non-membranous structures consisting of maternal RNAs and proteins required for the formation of germ cells. Drosophila germ plasm, also called pole plasm, forms at the posterior pole of the embryo and is inherited by the germline precursors, or pole cells. Because the cytoplasmic transplantation of the pole plasm into recipient embryos causes the ectopic formation of pole cells, the pole plasm contains sufficient factors for germ-cell formation. This observation also highlights the importance of retaining the pole plasm at the posterior cortex of the embryo to ensure the germ cells form at the appropriate location (Tanaka, 2008).

In Drosophila, the pole plasm is assembled during oogenesis, which is divided into 14 morphologically distinct stages of egg chamber development. The egg chamber is composed of a single oocyte and 15 nurse cells, surrounded by a monolayer of somatic follicle cells. During oogenesis, most components of pole plasm are synthesized in the nurse cells and transported into the oocyte via ring canals, which are cytoplasmic bridges interconnecting the oocyte with nurse cells. Within the oocyte, these factors become concentrated at the posterior pole and are assembled into the polar (germ) granules. These factors are transported by a polarized microtubule (MT) array that is initially nucleated at the oocyte posterior and extends into the nurse cells through the ring canals. During stages 6-7, the MT array is reorganized by the transforming growth factor alpha-like Gurken (Grk) signal. In the stage-6 oocyte, posteriorly restricted Grk induces neighboring follicle cells to adopt the posterior fate. These cells send back as-yet unknown signals to the oocyte to trigger the reorganization of the MT cytoskeleton. Consequently, the MT array within the oocyte becomes polarized along the anteroposterior (AP) axis, with the minus ends abundant at the anterior of the oocyte and the plus ends extending toward the posterior. This MT organization promotes the migration of the oocyte nucleus and associated grk RNA to the future anterior-dorsal corner, where Grk signals the follicle cells to define the dorsoventral axis. The polarized MT array also directs the localization of bicoid (bcd) RNA to the anterior and oskar (osk) RNA to the posterior within the oocyte. The anterior accumulation of bcd RNA is required for the proper development of the embryonic head and thoracic structures. The posterior localization of osk RNA is essential for the formation of the germ cells and abdomen (Tanaka, 2008).

osk RNA localization is tightly coupled to translational control: only the posteriorly localized osk message is translated. The localized Osk protein, in turn, recruits downstream components of the pole plasm, such as Vasa (Vas) and Tudor (Tud) proteins, and the nanos, germ cell-less and polar granule component RNAs. Misexpression of Osk at the anterior of the oocyte causes ectopic pole plasm assembly and the formation of germ cells at the new site, indicating that Osk organizes pole plasm assembly (Tanaka, 2008).

Although osk has no known alternatively spliced variants, the osk message produces two protein isoforms, long and short Osk, by translation from in-frame alternative start codons. Short Osk shares its entire sequence with the long isoform. Nevertheless, genetic evidence shows that the two Osk isoforms have distinct functions in the assembly of the pole plasm. Long Osk is required for all the components of the pole plasm, including Osk itself, to be anchored to the posterior cortex, preventing their diffusion into the cytoplasm. However, the mechanism by which long Osk retains pole plasm components at the posterior cortex remains unknown (Tanaka, 2008).

A recent immuno-electron microscopic study revealed that the two Osk isoforms localize to distinct organelles in the oocyte posterior: long Osk associates with endosomes and short Osk is concentrated in the polar granules (Vanzo, 2007). Long Osk also upregulates endocytosis, which occurs preferentially at the oocyte posterior (Vanzo, 2007). Therefore, the endocytic pathway may be involved in pole plasm assembly downstream of long Osk, although data are lacking to show that the association between long Osk and endosomes is functionally significant. Several reports have suggested that vesicular trafficking is involved in pole plasm assembly and germ cell formation. For example, in mutants for Rab11, which encodes a small GTPase involved in the recycling of endosomes, osk RNA fails to be transported to the oocyte posterior, instead forming aggregates close to the posterior. However, the defects in osk RNA localization in Rab11 mutants are thought to be an indirect consequence of the disrupted MT polarization (Tanaka, 2008).

This study shows that Drosophila Rabenosyn-5 (Rbsn-5), a Rab5 effector protein involved in the early endocytic pathway, is required for osk RNA localization and pole plasm assembly. Although the primary defect of the rbsn-5 mutation is, as in the Rab11 mutant, caused by the failure to maintain MT polarity, which secondarily affects osk RNA localization, evidence is provided that the endocytic pathway also functions downstream of Osk to anchor the pole plasm components to the oocyte cortex (Tanaka, 2008).

Vas is a reliable marker for the germline throughout Drosophila development. A GFP-Vas fusion protein enables the direct visualization of the pole plasm and germ cells in the living organism. During oogenesis, GFP-Vas accumulates at the oocyte posterior from stage 9 onward. Using GFP-Vas as a marker, a germline clonal screen was performed targeting chromosome 2L for mutations that disrupted pole plasm assembly. From 5122 lines mutagenized with EMS, 66 mutants were isolated defective in GFP-Vas localization. Twenty-seven of these were alleles of cappuccino, spire or profilin (chickadee), three genes on 2L that are known to be involved in osk RNA localization, which validates the screening strategy (Tanaka, 2008).

Among the other mutants recovered was a recessive lethal mutation, C241, that mapped to 28C2-29E2. Subsequent deficiency mapping and sequencing of the mutant chromosome revealed that the C241 mutation was a single nucleotide substitution in the CG8506 gene, which resulted in a premature stop codon at position 315 of the 505 amino acid open reading frame (ORF). The introduction of a transgene containing a genomic DNA fragment with the CG8506 transcriptional unit rescued the C241 mutant phenotypes (described below). These data show that CG8506 corresponds to the gene that was mutated at the C241 locus. Rabbit and rat polyclonal antisera raised against full-length CG8506 did not detect a truncated form of CG8506 in ovarian extracts from C241 heterozygotes. Furthermore, neither antibody showed immunoreactivity in C241 homozygous clones, suggesting that the truncated protein was not expressed at detectable levels and/or was unstable. Therefore, C241 appeared to be a strong loss-of-function, presumably a protein-null, allele of CG8506 (Tanaka, 2008).

CG8506 (Rabenosyn -- FlyBase) encodes a protein homologous to Rabenosyn-5 (Rbsn-5). Rbsn-5 interacts with several Rab proteins, including Rab5, which functions in early endosomal transport. Several Rbsn-5 protein domains are conserved across species, including the FYVE domain, which binds phosphatidylinositol-3-phosphate. However, invertebrate Rbsn-5 homologs lack the C-terminal domain common to the mammalian homologs of this protein. Since the C-terminal domain of mammalian Rbsn-5 is responsible for its interaction with Rab5, whether CG8506 interacted with Rab5 was examined. Pull-down assays showed that GST-Rab5 efficiently pulled down in-vitro-synthesized CG8506 protein in the presence of a GTP analog, GTP-γS, but inefficiently in the presence of GDP. The interaction between CG8506 and Rab5-GTP was specific, because the interactions of CG8506 with Rab11 and Rab7 were at background levels. Consistent with a physical interaction between CG8506 and Rab5 in vitro, in CG8506^C241 GLCs, neither auto-fluorescent granules derived from endocytosed yolk proteins nor the incorporation of a fluorescent marker for endocytosis, FM4-64, were observed in the oocytes, suggesting that CG8506 functions cooperatively with Rab5 in the early endocytic pathway. Thus, CG8506 is the Drosophila ortholog of Rbsn-5 and has an evolutionarily conserved function in the endocytic pathway (Tanaka, 2008).

This study shows that that Osk maintains, but does not establish, the posterior accumulation of endosomal proteins and asymmetric endocytosis, and that Osk can recruit endosomal proteins and stimulate endocytosis even at an ectopic site. It is further shown that the anchoring of the pole plasm components to the oocyte cortex requires the Osk-dependent stimulation of endocytic activity. These data reveal an interdependent relationship between Osk anchoring and localized endocytic activity at the oocyte posterior (Tanaka, 2008).

In rbsn-5^- oocytes, the anterior misexpression of Osk induces aberrant F-actin aggregates, which diffuse along with pole plasm components into the cytoplasm. Several lines of evidence suggest that the anchoring of pole plasm components requires the proper organization of F-actin. Since endosomal proteins are recruited by long Osk, the idea is favored that the endocytic pathway functions downstream of long Osk to anchor the pole plasm components at the cortex by regulating F-actin dynamics. Supporting this idea, in addition to its roles in early endosomal sorting, Rab5 acts as a signaling molecule that remodels F-actin networks. Rab11, which regulates the recycling of endosomes, is also involved in F-actin organization during cellularization in Drosophila blastoderm embryos. Intriguingly, the recruitment of endosomal proteins by Osk is not sufficient for proper F-actin reorganization to anchor the pole plasm components at the cortex, because their recruitment occurs even in oocytes lacking Rbsn-5, in which cortical anchoring fails. It is therefore proposed that the continuous cycling of endosomes is required for pole plasm components to be anchored to the oocyte cortex. This scenario is compatible with a model in yeasts, which use endocytic cycling coupled with localized exocytosis to maintain their polarity, although it is unclear if F-actin reorganization is involved in this process (Tanaka, 2008).

Rbsn-5 is primarily required for the maintenance of MT polarity that directs posterior localization of osk RNA. Rab11 is also required for MT polarization in the oocyte. However, the accumulation of endosomal proteins and upregulation of endocytosis at the oocyte posterior require the oocyte polarization, which promotes the reorganization of the MT array. Thus, MT polarization and asymmetric activation of the endocytic pathway are probably interdependent as well. Furthermore, maintenance of polarized endocytic activity depends on Osk. Intriguingly, Osk is also thought to maintain MT polarity, as posterior accumulation of Kin-βgal is partially defective in the absence of Osk (Zimyanin, 2007). It is therefore likely that the endocytic pathway and Osk form a positive-feedback loop that maintains oocyte polarity: Osk may maintain MT polarity through recruiting endosomal proteins. Based on these results, a model is proposed in which the endocytic pathway is involved in several distinct steps in pole plasm assembly (Tanaka, 2008).

The localization of bcd RNA to the anterior pole of the oocyte requires the ESCRT-II (endosomal sorting complex required for transport II) complex, which sorts mono-ubiquitinated endosomal transmembrane proteins into multivesicular bodies. Furthermore, Vps36p, a component of the ESCRT-II complex, binds bcd 3' UTR in vitro and co-localizes with bcd RNA at the oocyte anterior, suggesting the direct involvement of ESCRT-II in bcd RNA localization. osk RNA, however, appears to use another mechanism for its posterior localization, since its localization is unaffected in the absence of ESCRT-II function. Several lines of evidence suggest that ER organization and RNA localization are linked. However, it is considered unlikely that the ER directs the posterior localization of osk RNA, because ER components and osk RNP distributed differentially in developing oocytes. Interestingly, the osk RNP and the endosomal proteins are in close proximity during their transport to the oocyte posterior. Although their close association may simply be owing to the dynamic rearrangements of the MT array during stages 7-8, these findings suggest that the endocytic pathway may also play a role in the targeting of osk RNP to the posterior pole of the oocyte. Retroviral genomic RNAs are known to hitchhike on endosomal vesicles to reach the plasma membrane. Therefore, it will be interesting to learn if osk RNA is also transported to the posterior pole of the oocyte along with the endosomes (Tanaka, 2008).

Search PubMed for articles about Drosophila Rabenosyn

Abe, M., et al. (2009). Membrane protein location-dependent regulation by PI3K (III) and rabenosyn-5 in Drosophila wing cells. PLoS ONE 4: e7306. PubMed ID: 19798413

Bryant, N. J., Piper, R. C., Gerrard, S. R. and Stevens, T. H. (1998). Traffic into the prevacuolar/endosomal compartment of Saccharomyces cerevisiae: a VPS45-dependent intracellular route and a VPS45-independent, endocytic route. Eur. J. Cell Biol. 76: 43-52. PubMed ID: 9650782

Cowles, C. R., Emr, S. D. and Horazdovsky, B. F. (1994). Mutations in the VPS45 gene, a SEC1 homologue, result in vacuolar protein sorting defects and accumulation of membrane vesicles. J. Cell Sci. 107: 3449-3459. PubMed ID: 7706396

De Renzis, S., Sonnichsen, B. and Zerial, M. (2002). Divalent Rab effectors regulate the sub-compartmental organization and sorting of early endosomes. Nat Cell Biol 4: 124-133. PubMed ID: 11788822

Eathiraj, S., Pan, X., Ritacco, C. and Lambright, D. G. (2005). Structural basis of family-wide Rab GTPase recognition by rabenosyn-5. Nature 436: 415-419. PubMed ID: 16034420

Fasshauer, D., Sutton, R.B., Brunger, A.T., and Jahn, R. (1998). Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q- and R-SNAREs. Proc. Natl. Acad. Sci. 95: 15781-15786. PubMed ID: 9861047

Gengyo-Ando, K., Kuroyanagi, H., Kobayashi, T., Murate, M., Fujimoto, K., Okabe, S. and Mitani, S. (2007). The SM protein VPS-45 is required for RAB-5-dependent endocytic transport in Caenorhabditis elegans. EMBO Rep. 8: 152-157. PubMed ID: 17235359

Grosshans, B. L., Ortiz, D. and Novick, P. (2006). Rabs and their effectors: Achieving specificity in membrane traffic. Proc. Natl. Acad. Sci. 103: 11821-11827. PubMed ID: 16882731

Lu, H. and Bilder, D. (2005). Endocytic control of epithelial polarity and proliferation in Drosophila. Nat. Cell Biol. 7: 1232-1239. PubMed ID: 16258546

McBride, H. M., Rybin, V., Murphy, C., Giner, A., Teasdale, R. and Zerial, M. (1999). Oligomeric complexes link Rab5 effectors with NSF and drive membrane fusion via interactions between EEA1 and Syntaxin 13. Cell 98: 377-386. PubMed ID: 10458612

Menut, L., Vaccari, T., Dionne, H., Hill, J., Wu, G. and Bilder, D. (2007). A mosaic genetic screen for Drosophila neoplastic tumor suppressor genes based on defective pupation. Genetics 177: 1667-1677. PubMed ID: 17947427

Morrison, H. A., Dionne, H., Rusten, T. E., Brech, A., Fisher, W. W., Pfeiffer, B. D., Celniker, S. E., Stenmark, H. and Bilder, D. (2008). Regulation of early endosomal entry by the Drosophila tumor suppressors Rabenosyn and Vps45. Mol. Biol. Cell 19(10): 4167-76. PubMed ID: 18685079

Mottola, G., et al. (2010). A novel function for the Rab5 effector Rabenosyn-5 in planar cell polarity. Development 137(14): 2353-64. PubMed ID: 20534670

Naslavsky, N., Boehm, M., Backlund, P. S. and Caplan, S. (2004). Rabenosyn-5 and EHD1 interact and sequentially regulate protein recycling to the plasma membrane. Mol. Biol. Cell 15: 2410-2422. PubMed ID: 15020713

Nichols, B. J., Holthuis, J. C. and Pelham, H. R. (1998). The Sec1p homologue Vps45p binds to the syntaxin Tlg2p. Eur. J. Cell Biol. 77: 263-268. PubMed ID: 9930650

Nielsen, E., Christoforidis, S., Uttenweiler-Joseph, S., Miaczynska, M., Dewitte, F., Wilm, M., Hoflack, B. and Zerial, M. (2000). Rabenosyn-5, a novel Rab5 effector, is complexed with hVPS45 and recruited to endosomes through a FYVE finger domain. J. Cell Biol. 151: 601-612. PubMed ID: 11062261

Peterson, M. R., Burd, C. G. and Emr, S. D. (1999). Vac1p coordinates Rab and phosphatidylinositol 3-kinase signaling in Vps45p-dependent vesicle docking/fusion at the endosome. Curr. Biol. 9: 159-162. PubMed ID: 10021387

Raymond, C. K., Howald-Stevenson, I., Vater, C. A. and Stevens, T. H. (1992). Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants. Mol. Biol. Cell 3: 1389-1402. PubMed ID: 1493335

Rubino, M., Miaczynska, M., Lippe, R. and Zerial, M. (2000). Selective membrane recruitment of EEA1 suggests a role in directional transport of clathrin-coated vesicles to early endosomes. J. Biol. Chem. 275: 3745-3748. PubMed ID: 10660521

Stenmark, H. and Olkkonen, V. M. (2001). The Rab GTPase family. Genome Biol. 2(5):REVIEWS3007. PubMed ID: 11387043

Tanaka, T. and Nakamura, A. (2008). The endocytic pathway acts downstream of Oskar in Drosophila germ plasm assembly. Development 135: 1107-1117. PubMed ID: 18272590

Vaccari, T. and Bilder, D. (2005). The Drosophila tumor suppressor vps25 prevents nonautonomous overproliferation by regulating notch trafficking. Dev. Cell 9: 687-698. PubMed ID: 16256743

Vaccari, T., Lu, H., Kanwar, R., Fortini, M. E. and Bilder, D. (2008). Endosomal entry regulates Notch receptor activation in Drosophila melanogaster. J. Cell Biol. 180: 755-762. PubMed ID: 18299346

Vanzo, N., Oprins, A., Xanthakis, D., Ephrussi, A. and Rabouille, C. (2007). Stimulation of endocytosis and actin dynamics by Oskar polarizes the Drosophila oocyte. Dev. Cell 12: 543-555. PubMed ID: 17419993

Wucherpfennig, T., Wilsch-Brauninger, M. and Gonzalez-Gaitan, M. (2003). Role of Drosophila Rab5 during endosomal trafficking at the synapse and evoked neurotransmitter release. J. Cell Biol. 161: 609-624. PubMed ID: 12743108

Zerial, M. and McBride, H.M. (2001). Rab proteins as membrane organizers. Nat. Rev. Mol. Cell Biol. 2: 107-117. PubMed ID: 11252952

Zimyanin, V., Lowe, N. and St Johnston, D. (2007). An Oskar-dependent positive feedback loop maintains the polarity of the Drosophila oocyte. Curr. Biol. 17(4): 353-9. PubMed ID: 17275299

date revised: 15 December 2010

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