Gene name - Rab11
Cytological map position - 93B12--13
Function - signaling
Symbol - Rab11
FlyBase ID: FBgn0015790
Genetic map position - 3R
Classification - GTP-binding protein domain
Cellular location - cytoplasmic
|Recent literature||Ji, H. H., Zhang, H. M., Shen, M., Yao, L. L. and Li, X. D. (2015). The motor function of Drosophila melanogaster myosin-5 is activated by calcium and cargo-binding protein dRab11. Biochem J [Epub ahead of print]. PubMed ID: 25940004
Myosin-5 (DmM5; Didum) plays two distinct roles in response to light stimulation: transport of pigment granules to the rhabdomere base to decrease light exposure and transport of rhodopsin-bearing vesicles to the rhabdomere base to compensate for the rhodopsin loss during light exposure. This study overexpressed DmM5 in Sf9 cells and investigated its regulation using purified proteins. The actin-activated ATPase activity of DmM5 is significantly lower than that of the truncated DmM5 having the C-terminal globular tail domain (GTD) deleted, indicating that the GTD is the inhibitory domain. The actin-activated ATPase activity of DmM5 is significantly activated by micromolar levels of calcium. DmM5 associates with pigment granules and rhodopsin-bearing vesicles through cargo-binding proteins Lightoid and dRab11 respectively. GTP-bound dRab11, but not Lightoid, significantly activates DmM5 actin-activated ATPase activity. Moreover, Gln1689 in the GTD was identified as the critical residue for the interaction with dRab11. Based on those results, it is proposed that DmM5-dependent transport of pigment granules is directly activated by light-induced calcium influx and the DmM5-dependent transport of rhodopsin-bearing vesicle is activated by active GTP-bound dRab11, whose formation is stimulated by light-induced calcium influx.
|Mavor, L. M., Miao, H., Zuo, Z., Holly, R. M., Xie, Y., Loerke, D. and Blankenship, J. T. (2016). Rab8 directs furrow ingression and membrane addition during epithelial formation in Drosophila melanogaster. Development [Epub ahead of print]. PubMed ID: 26839362
One of the most fundamental changes in cell morphology is the ingression of a plasma membrane furrow. The Drosophila embryo undergoes several cycles of rapid furrow ingression during early development that culminates in the formation of an epithelial sheet. Previous studies have demonstrated the requirement for intracellular trafficking pathways in furrow ingression; however, the pathways that link compartmental behaviors with cortical furrow ingression events have remained unclear. This study shows that Rab8 has striking dynamic behaviors in vivo. As furrows ingress, cytoplasmic Rab8 puncta are depleted and Rab8 accumulates at the plasma membrane in a location that coincides with known regions of directed membrane addition. CRISPR/Cas9 technology was used to N-terminally tag Rab8, which is then used to address both endogenous localization and function. Endogenous Rab8 displays partial coincidence with Rab11 and the Golgi, and this colocalization is enriched during the fast phase of cellularization. When Rab8 function is disrupted, furrow formation in the early embryo is completely abolished. Rab8 behaviors require the function of the exocyst complex subunit Sec5 as well as the recycling endosome Rab11. Active, GTP-locked Rab8 is primarily associated with dynamic membrane compartments and the plasma membrane, while GDP-locked Rab8 forms large cytoplasmic aggregates. These studies suggest a model in which active Rab8 populations direct furrow ingression by guiding the targeted delivery of cytoplasmic membrane stores to the cell surface through exocyst tethering complex interactions.
|Woichansky, I., Beretta, C. A., Berns, N. and Riechmann, V. (2016). Three mechanisms control E-cadherin localization to the zonula adherens. Nat Commun 7: 10834. PubMed ID: 26960923
E-cadherin localization to the zonula adherens is fundamental for epithelial differentiation but the mechanisms controlling localization are unclear. Using the Drosophila follicular epithelium, E-cadherin transport was genetically dissected in an in vivo model. Three mechanisms were distinguished mediating E-cadherin accumulation at the zonula adherens. Two membrane trafficking pathways deliver newly synthesized E-cadherin to the plasma membrane. One is Rab11 dependent and targets E-cadherin directly to the zonula adherens, while the other transports E-cadherin to the lateral membrane. Lateral E-cadherin reaches the zonula adherens by endocytosis and targeted recycling. This pathway is dependent on RabX1, which provides a functional link between early and recycling endosomes. Moreover, lateral E-cadherin is transported to the zonula adherens by an apically directed flow within the plasma membrane. Differential activation of these pathways could facilitate cell shape changes during morphogenesis, while their misregulation compromises cell adhesion and tissue architecture in differentiated epithelia.
|Sorvina, A., Shandala, T. and Brooks, D. A. (2016). Drosophila Pkaap regulates Rab4/Rab11-dependent traffic and Rab11 exocytosis of innate immune cargo. Biol Open [Epub ahead of print]. PubMed ID: 27190105
The secretion of immune-mediators is a critical step in the host innate immune response to pathogen invasion, and Rab GTPases have an important role in the regulation of this process. Rab4/Rab11 recycling endosomes are involved in the sorting of immune-mediators into specialist Rab11 vesicles that can traffic this cargo to the plasma membrane; however, how this sequential delivery process is regulated has yet to be fully defined. This study reports that Drosophila Pkaap, an orthologue of the human dual-specific A-kinase-anchoring protein 2 or D-AKAP2 (also called AKAP10), appeared to have a nucleotide-dependent localisation to Rab4 and Rab11 endosomes. RNAi silencing of pkaap altered Rab4/Rab11 recycling endosome morphology, suggesting that Pkaap functions in cargo sorting and delivery in the secretory pathway. The depletion of pkaap also had a direct effect on Rab11 vesicle exocytosis and the secretion of the antimicrobial peptide Drosomycin at the plasma membrane. It is proposed that Pkaap has a dual role in antimicrobial peptide traffic and exocytosis, making it an essential component for the secretion of inflammatory mediators and the defence of the host against pathogens.
|Catrina, I. E., Bayer, L. V., Yanez, G., McLaughlin, J. M., Malaczek, K., Bagaeva, E., Marras, S. A. and Bratu, D. P. (2016). The temporally controlled expression of Drongo, the fruit fly homolog of AGFG1, is achieved in female germline cells via P-bodies and its localization requires functional Rab11. RNA Biol: 1-16. PubMed ID: 27654348
To achieve proper RNA transport and localization, RNA viruses exploit cellular vesicular trafficking pathways. AGFG1, a host protein essential for HIV-1 and Influenza A replication, has been shown to mediate release of intron-containing viral RNAs from the perinuclear region. It is still unknown what its precise role in this release is, or whether AGFG1 also participates in cytoplasmic transport. This study reports for the first time the expression patterns during oogenesis for the Arf GTPase activating protein Drongo, the fruit fly homolog of AGFG1. Temporally controlled Drongo expression is achieved by translational repression of drongo mRNA within P-bodies. This study shows a first link between the recycling endosome pathway and Drongo and finds that proper Drongo localization at the oocyte's cortex during mid-oogenesis requires functional Rab11.
In developing Drosophila photoreceptors, rhodopsin is trafficked to the rhabdomere, a specialized domain within the apical membrane surface. Rab11, a small GTPase implicated in membrane traffic, immunolocalizes to the trans-Golgi network, cytoplasmic vesicles and tubules, and the base of rhabdomeres. One hour after release from the endoplasmic reticulum, rhodopsin colocalizes with Rab11 in vesicles at the base of the rhabdomere. When Rab11 activity is reduced by three different genetic procedures, rhabdomere morphogenesis is inhibited and rhodopsin-bearing vesicles proliferate within the cytosol. Rab11 activity is also essential for development of multivesicular body (MVB) endosomal compartments; this is probably a secondary consequence of impaired rhabdomere development. Furthermore, Rab11 is required for transport of TRP, another rhabdomeric protein, and for development of specialized membrane structures within Garland cells. These results establish a role for Rab11 in the post-Golgi transport of rhodopsin and of other proteins to the rhabdomeric membranes of photoreceptors, and in analogous transport processes in other cells (Satoh, 2005).
Rab11 also functions during oogenesis and during cellularization of Drosophila embryos. The Nuclear fallout and Rab11 function in membrane trafficking and actin remodeling during the initial stages of furrow formation during cellularization. Membrane addition is mediated via endosomal-mediated membrane delivery to the site of furrow formation. Thus Rab11 regulates endosomes as key trafficking intermediates that control vesicle exocytosis and membrane growth during cellularization (Riggs, 2003; Pelissier, 2003). Rab11 is required in endocytic recycling and in the organization of posterior membrane compartments during oogenesis. Rab11 is also required in the organization of microtubule plus ends and osk mRNA localization and translation at the posterior pole (Jankovics, 2001). It is proposed that microtubule plus ends and, possibly, translation factors for osk mRNA are anchored to posterior membrane compartments that are defined by Rab11-mediated trafficking and reinforced by Rab11-Osk interactions (Dollar, 2002).
During photoreceptor terminal differentiation, massive biosynthetic membrane traffic delivers rhodopsin and other phototransduction proteins to an apical plasma membrane subdomain to form photosensory organelles, invertebrate rhabdomeres and vertebrate outer segments. The proper targeting of rhodopsin to this domain is crucial for normal development and, if impaired, leads to retinal degeneration. Rab proteins and their effectors are known to control membrane traffic and maintain distinct organelle identities (Deneka, 2003). Indeed, observations in Xenopus (Deretic, 1995; Moritz, 2001) and Drosophila (Satoh, 1997; Shetty, 1998) support a role for particular Rab proteins in rhodopsin transport to the photosensitive organelles (Satoh, 2005 and references therein).
Surprisingly, these studies on rhodopsin trafficking identified Rab11 as active in exocytosis. Rab11 is thought to regulate endosomal/plasma membrane interactions by controlling membrane traffic through recycling endosomes. These endosomes receive endocytosed plasma membrane and either return it to the cell surface or direct it to degradative pathways. Rab11 localizes to the pericentriolar recycling endosome, the trans-Golgi network (TGN), and post-Golgi vesicles (Chen, 1998; Deretic, 1997; Ullrich, 1996). Dominant-negative Rab11a inhibits apical recycling and basolateral-to-apical transcytosis in polarized MDCK cells (Wang, 2000), blocks stimulus-induced recruitment of endosome-sequestered H+-K+ ATPase-rich membrane to the apical membrane of acid-secreting parietal cells (Duman, 1999), and inhibits exosome release in human leukemic K562 cells (Savina, 2002; Satoh, 2005 and references therein).
In addition to these extensive reports linking Rab11 activity to endocytic pathways, other reports suggest a role for Rab11 in biosynthetic exocytic membrane traffic. In PC12 cells, Rab11 was detected in association with TGN and TGN-derived secretory vesicles (Urbe, 1993). In baby hamster kidney cells, overexpression of dominant-negative Rab11S25N decreased delivery of the basolaterally targeted vesicular stomatitis virus (VSV) G protein to the cell surface (Chen, 1998), and expression of wild-type Rab11a accelerated delivery of new protease activated receptors to kidney epithelial cell surfaces following trypsin exposure (Roosterman, 2003). Of particular interest to this study, the movement of rhodopsin to the apical surface may also be dependent on Rab11. Rhodopsin has been detected in Rab11-positive post-Golgi vesicles of Xenopus retina cell-free extracts (Deretic, 1997). Immature rhodopsin, which is indicative of defective rhodopsin transport, accumulated in Drosophila photoreceptors that expressed dominant-negative Rab11N124I (Satoh, 1998; Satoh, 2005 and references therein).
The movement of rhodopsin and other rhabdomeric membrane proteins has been characterized in the developing Drosophila photoreceptor. This experimental system allows the role of Rab11 in this process to be defined. Vigorous light-dependent endocytosis competes with exocytosis from the outset of rhabdomere morphogenesis. Independent of a requirement in endosomal recycling, Rab11 activity is essential for the initial exocytic rhodopsin delivery to the growing rhabdomere. Loss of Rab11 activity disrupts endocytic pathways, but this is likely to be a secondary consequence of attenuated exocytic delivery. Thus, these results demonstrate Rab11 promotes the trans-Golgi to rhabdomere membrane traffic responsible for elaboration of the sensory membranes of these cells (Satoh, 2005).
Drosophila rhabdomere differentiation begins in mid-pupal life with the establishment of a morphologically and molecularly distinct apical plasma membrane subdomain, which is then amplified and specialized for phototransduction by targeted membrane delivery (Karagiosis, 2004; Longley, 1995). Between ~50% and 60% of pupal development (% pd), nascent rhabdomeres begin to load with TRP, a light-activated Ca2+ channel that serves phototransduction. TRP is first immunodetectable in photoreceptor cytoplasm beginning at about 45% pd and accumulates in large cytoplasmic vesicles by 50% pd. Expression of the major rhodopsin (Rh1), the photosensory protein of photoreceptors R1-6, initiates later, at about 70% pd. Rh1 is first detected as faint diffuse signals and small puncta spread throughout the cytoplasm. In animals raised in standard 12/12 hour light/dark conditions, Rh1 concentrates in large (>200 nm) cytoplasmic vesicles, 'Rh1-containing large vesicles' (RLVs), prior to its appearance in the rhabdomere. During the stage when Rh1 and TRP synthesis overlap, the proteins colocalize in RLVs, suggesting the same vesicle accommodates both rhabdomere proteins. One or more small dots of F-actin decorate each RLV, resembling the actin patches associated with vesicles that mediate transport in yeast and other systems. In both fixed and living preparations, RLVs appear tethered via actin patches to the rhabdomere terminal web (RTW), a specialization of the cortical actin cytoskeleton (Satoh, 2005).
Immunogold electron microscopy using anti-Rh1 identified RLVs as multivesicular bodies (MVBs). The delicate detail of MVBs is poorly preserved by fixation protocols that retain Rh1 antigenicity. However, MVBs are the only organelle observed in the EM with the size, shape and distribution characteristic of RLVs observed in confocal immunofluorescence studies. RLVs label with GFP-tagged Rab7, an endosomal protein that marks the limiting membrane of MVBs in Drosophila. RLVs also immunostain for Hrs, the endosome-associated Hepatocyte growth factor-regulated tyrosine kinase substrate associated with MVBs in Drosophila and other systems. These observations identify RLVs as MVBs (Satoh, 2005).
The early detection of rhodopsin and TRP within MVBs was not anticipated because MVBs are generally considered to be late endosomal compartments, delivering cargo retrieved from the plasma membrane to the lysosomes for degradation. Thus, the presence of Rh1 in an endocytic degradative organelle during the time the cell is increasing its sensory membrane is noteworthy. However, light-dependent endocytosis of Rh1 is well documented in adult photoreceptors, and it is possible that the Rh1 found in RLVs has already been retrieved from the developing rhabdomere (Satoh, 2005).
To investigate this possibility, Rh1 transport was observed in dark-reared flies. Rh1 begins to accumulate in the rhabdomere just after Rh1 expression starts at 70% pd. There are few RLVs in all stages in dark-reared flies. RLVs form within 30 minutes of light exposure, and disappear within 13 hours of return to dark. These observations suggest that in light-reared flies, Rh1 is first transported to the rhabdomere, but light-induced internalization quickly transports Rh1 into RLVs. Thus, even during the developmental period in which the photoreceptor cell is increasing rhabdomeric volume and Rh1 content, vigorous endocytosis can exceed the rate of biosynthetic delivery of Rh1 (Satoh, 2005).
To position Rab11 in the sensory membrane transport pathway, flies expressing the Golgi marker, CFP-galactosyl-transferase and Rab11 were immunolocalized in developing photoreceptors. Rab11 is present on small vesicles (<200 nm in diameter) scattered throughout the cytoplasm. Many of these appear associated with Golgi structures; others are located at the base of developing rhabdomere. Rab11 is not associated with RLVs. To further characterize the Golgi association of Rab11, developing photoreceptors were immunostained for Rab11 and the cis-Golgi marker, Rab1. Rab1 localizes to the convex side of the Golgi, while Rab11 localizes to the opposite, concave, side. Triple staining of CFP-labeled Golgi directly shows Rab1 and Rab11 localize to opposite sides of the Golgi. Therefore, Rab11 must localize to the trans-Golgi surface (Satoh, 2005).
To investigate the possibility that Rab11 is associated with maturing Rh1, blue light was used to trigger synchronized release of Rh1 accumulated in the ER because of lack of the correct chromophore isomer. Rh1 and Rab11 were then immunolocalized in pupal eyes 0, 40, 60, 90 and 180 minutes after blue-light irradiation. Prior to blue light (0 minutes), Rh1 was distributed throughout photoreceptor cytoplasm, colocalizing with an ER marker. At 40 and 60 minutes, most Rh1 colocalized with the Golgi. By 40 minutes, some Rh1 was concentrated in Rab11 immunopositive vesicles, some of these at the rhabdomere base. At both 60 and 90 minutes, Rh1 and Rab11 show extensive colocalization in vesicles at the rhabdomere base. By 90 minutes, there are few Rh1 positive cytoplasmic vesicles, and by 180 minutes most Rh1 is found in the rhabdomeres (Satoh, 2005).
These immunofluorescence results suggest Rh1, upon exit from the ER, associates first with the Golgi, then within Rab11-positive vesicles, before being deposited in the rhabdomere. Thus, the Rab11 localization is consistent with a role in TGN--->rhabdomere transport. Transport visualized in these studies is completed by 180 minutes, in good agreement with previous immunoblot data showing intermediate Rh1 is completely processed into mature 35K Rh1 within 180 minutes (Satoh, 1997). RLVs are not prominent at any time during ER--->rhabdomere transport, consistent with the proposal that RLVs do not participate in biosynthetic traffic (Satoh, 2005).
Thus Rab11 is essential for trafficking two membrane proteins, TRP and Rh1, to the Drosophila photosensory organelle, the rhabdomere. When Rab11 activity is reduced in developing photoreceptors, Golgi-derived TRP- and Rh1-bearing vesicles accumulate in photoreceptor cytoplasm instead of exocytosing to expand the growing rhabdomere. Thus Rab11 activity supports a distinct plasma membrane compartment, the apical plasma membrane subdomain specialized for phototransduction (Satoh, 2005).
Rab11 has been implicated in control of membrane traffic through the pericentriolar recycling endosome. In cultured baby hamster kidney (BHK) cells, return of internalized transferrin receptor to the cell surface is inhibited by dominant-negative Rab11 expression (Ullrich, 1996). During cellularization of Drosophila embryos, apical membrane redeployed to the growing basolateral surface transits a Rab11-dependent recycling endosome (Pelissier, 2003). Rab11 has also been implicated in trans-Golgi to plasma membrane transport. In non-polarized BHK cells in culture, expression of dominant-negative Rab11S25N inhibits transport of a basolateral marker protein marker, vesicular stomatitis virus G protein, but has no impact on delivery of an apical marker protein, influenza hemagglutinin (Chen, 1998). Recent observation that recycling endosomes can serve as an intermediate during transport from the Golgi to MDCK cell plasma membranes raises the possibility that biosynthetic traffic transits a recycling endosome and the site of Rab11 action is at the recycling endosome. However, no pericentriolar endosome has been observed in Drosophila photoreceptors, and Rh1 moves directly from the trans-Golgi to the rhabdomere when released into the biosynthetic pathway by blue light. Thus, there is no evidence for Rh1 moving through an intermediate compartment when en route to the rhabdomere (Satoh, 2005).
Rh1-bearing post-Golgi vesicles and recycling endosome-derived vesicles may both traffick to the cell surface because they share common Rab11 effectors. Rab11 interacts with unconventional class V myosins and expression of dominant-negative Myo-Vb inhibits delivery from early endosomes to the cell surface (Lapierre, 2001). An extensive F-actin terminal web, the RTW, extends from the rhabdomere base into photoreceptor cell cytoplasm (Chang, 2000) and disruption of the photoreceptor actin cytoskeleton inhibits the vesicular traffic that builds crab rhabdomeres. Rab11, together with a Myo-V effector, may promote post-Golgi vesicle motility along the actin RTW to focus delivery to the rhabdomere (Satoh, 2005).
Loss of Rab11 activity also disrupts normal photoreceptor MVB morphology. MVBs are often identified as late endosomal compartments, delivering cargo destined for lysosomal degradation. However, several recent studies show MVBs can be also exocytic carriers, delivering endosomal contents to the cell surface. Examples include the secretory lysosomes of immune system cells, melanosomes of pigment cells, exosomes of maturing red blood cells and secreted vesicles mediating cell-cell signaling. The accumulation of newly synthesized MHCII receptors within MVBs of unstimulated dendritic cells, and the stimulus-induced reorganization of MVBs and appearance of MHCII receptors at the plasma membrane, has led to consideration of MVBs as an exocytic compartment. Autoradiography of crayfish eyes following 3H-leucine injection has shown newly synthesized protein first in the cytoplasm, then in MVBs and then in rhabdomere rhabdomeres, prompting the conjecture that MVBs are a post-Golgi organelle of biosynthetic traffic (Satoh, 2005).
The work reported in this study discounts the possibility that MVBs are exocytic vesicles in Drosophila photoreceptors. It was shown that appearance of Rh1 in the MVBs is dependent on light treatment. Previously, light was shown to trigger endocytosis of rhabdomeric membrane, so this light dependency suggests MVBs originate from an endocytic process. Depletion of Rab5 activity, which is known to regulate the fusion between endocytic vesicles and early endosomes, also eliminates Rh1 and TRP containing MVBs, without affecting Rh1 and TRP transport to the rhabdomere. Thus, all the results support the view that MVBs are endocytic vesicles. The early and rapid appearance of Rh1 and TRP in these vesicles is remarkable, showing that the machinery of light-dependent receptor internalization is fully operational at the outset of morphogenesis. Vigorous light-dependent endocytosis competes with exocytosis from the outset of rhabdomere morphogenesis, internalizing rhodopsin and TRP from the growing sensory membrane even as exocytosis expands it (Satoh, 2005).
Rab5 loss-of-function analysis also supports the view that Rab11 acts before Rab5. In Rab5, Rab11 double mutants, photoreceptors retain the Rab11 phenotype. These results are consistent with a role of Rab11 in the exocytic process, but not with an exclusive role in endocytic recycling. Yet, Rab11 activity is required for accumulation of MVBs. It is proposed that this is an indirect effect of the Rab11 requirement in the exocytic pathway. Rab11 inhibition 'starves' the rhabdomere, the target of Rab11-mediated transport, of required proteins, which in turn slows the rate of endocytosis and eliminates endocytosis-dependent MVBs. In support of this view, Rab11 activity was shown to be required for the presence of labyrinthine channels on Garland cells, membrane specializations that promote vigorous endocytosis. Rab11 loss plausibly depletes membrane components that sustain vigorous endocytosis (Satoh, 2005).
Drosophila and vertebrate photoreceptors share fundamental cellular and molecular mechanisms and Rab family members and their functions are strongly conserved across eukaryotes. Rab11 has been identified in rhodopsin-containing post-Golgi vesicles formed within a vertebrate retina cell-free system (Deretic, 1997), raising the likelihood vertebrate photoreceptors also contain a Rab11-dependent vesicular compartment essential for rhodopsin transport and outer segment development. Failure to traffic Rh1 in Drosophila leads to retinal degeneration, and similar mechanisms are implicated in rhodopsin mutations and other mutations causing the human disease retinitis pigmentosa. The involvement of Rab11 in the post-Golgi processes provides an entry point to discover the cellular components and pathways responsible for elaborating the specialized photosensitive membranes. These events are likely to be key regulators of normal cellular development and the triggering events of retinal disease (Satoh, 2005).
Rhodopsins (Rhs) are light sensors, and Rh1 is the major Rh in the Drosophila photoreceptor rhabdomere membrane. Upon photoactivation, a fraction of Rh1 is internalized and degraded, but it remains unclear how the rhabdomeric Rh1 pool is replenished and what molecular players are involved. This study shows that Crag (Calmodulin-binding protein related to a Rab3 GDP/GTP exchange protein), a DENN protein, is a guanine nucleotide exchange factor for Rab11 that is required for the homeostasis of Rh1 upon light exposure. The absence of Crag causes a light-induced accumulation of cytoplasmic Rh1, and loss of Crag or Rab11 leads to a similar photoreceptor degeneration in adult flies. Furthermore, the defects associated with loss of Crag can be partially rescued with a constitutive active form of Rab11. It is proposed that upon light stimulation, Crag is required for trafficking of Rh from the trans-Golgi network to rhabdomere membranes via a Rab11-dependent vesicular transport (Xiong, 2012).
This study shows that Crag is a novel GEF for Rab11 and that it is required for the post-Golgi transport of Rh1 to the rhabdomeres during light activation. This regulated transport of Rh1, which is independent of Rh1 transport during the development of the photoreceptors, replenishes the loss of Rh1 induced by light stimulation. Loss of Crag leads to accumulation of secretory vesicles in the cytosol of photoreceptor cells, and eventually leads to a light- and age-dependent photoreceptor degeneration (Xiong, 2012).
During development of photoreceptors, Rh1 and other phototransduction proteins are synthesized in the endoplasmic reticulum and transported to the rhabdomeres to build functional photoreceptors. Some molecular players, including Rab11 and exit protein of rhodopsin and TRP (XPORT), have been shown to play a role in this process. Upon light activation Rh1 is converted to metaRh. MetaRh is then converted back into Rh1 on rhabdomere membranes via absorption of another photon, allowing the maintenance of Rh1 levels in the rhabdomere. In wild-type photoreceptors, a portion of metaRh is phosphorylated and endocytosed, and it has been proposed that internalization of metaRh promotes the clearance of dysfunctional proteins and serves as a proofreading mechanism. Internalized Rh1 is then degraded through an endosomal/lysosomal pathway. Obviously, the gradual loss of Rh1 in wild-type photoreceptors leads to the necessity to constitutively synthesize Rh1 and replenish the rhabdomeric pool. This is nicely illustrated with the loss of retinol dehydrogenase (RDH), which is required for the regeneration of the chromophore of Rh1. Loss of RDH leads to progressive reduction in rhabdomere size and light-dependent photoreceptor degeneration (Xiong, 2012).
The data show that Crag is required to maintain homeostasis of Rh1 upon light stimulation. Loss of Crag leads to Rh1 accumulation in the cytosol and, eventually, retinal degeneration in the presence of light. Mutations in genes that affect metaRh1 turnover, such as Calmodulin and arrestin 2, lead to prolonged deactivation time of the photoresponse. Since both ERGs and single-cell recordings of Crag mutant photoreceptors are normal, it is unlikely that Crag is involved in the recycling of metaRh1 to Rh1. To test whether Crag is required for transport of newly synthesized Rh1 in adult photoreceptors, flies exposed to blue light to trigger massive endocytosis and degradation of Rh1, and then the new synthesis and transport of Rh1 back to the rhabdomeres was measured over time. Crag is not required for the synthesis of Rh1. However, in Crag mutants, the newly synthesized Rh1 accumulates in the cytosol. It is proposed that Crag is required for the delivery of newly synthesized Rh1 to the rhabdomeres and that loss of Crag leads to a gradual reduction in the size of rhabdomeres and to degeneration of the photoreceptor cells. Indeed, the time course and morphological features of degeneration associated with loss of Crag are very similar to the phenotypes observed in RDH mutants, further supporting that Crag is involved in the Rh1 synthesis/delivery pathway (Xiong, 2012).
Rab11 has been implicated in various intracellular membrane trafficking processes. Its diverse functions in different membrane compartments are mediated through its downstream effectors in a context-specific manner; many of these functions have been identified in previous studies. However, GEFs for Rab11 in any context have not yet been identified. In vivo and in vitro data provide compelling evidence that Crag is a GEF for Rab11. First, in Drosophila S2 cells, Crag colocalizes and physically interacts with Rab11. Second, Crag preferably binds to the GDP-bound form of Rab11, and the DENN domains are required for binding. Third, Crag is required for the proper localization of Rab11 in photoreceptors upon light stimulation. Fourth, loss of Crag or Rab11 leads to a similar light-induced photoreceptor degeneration. Fifth, expression of Rab11-CA partially rescues the degeneration caused by Crag mutations. Finally, an in vitro GEF assay shows that Crag facilitates the release of GDP from Rab11. It has been previously established that Rab11 is essential for photoreceptor cell development and Rh1 transport during pupal stages. However both rhabdomere morphology and Rh1 localization are normal in Crag clones in newly eclosed flies. Similarly, initial deposition of TRP is also not affected by Crag mutations, in agreement with previous findings that Rh1 and TRP are co-transported to the rhabdomeres during their development. Interestingly, cytosolic localization of TRP is not observed in Crag mutant photoreceptor cells exposed to light, suggesting that during light stimulation, Rh1 and TRP dynamics are distinct. Indeed, internalization of TRP upon light stimulation has not been reported in previous studies. The current data therefore indicate that other GEFs must exist for Rab11 during photoreceptor development, and that Crag is specifically required for Rab11 GDP/GTP exchange during light activation in adult flies. In addition, Crag may function as a GEF for Rab10 in other processes and cells, such as polarized deposition of basement membrane proteins in follicle cells (Xiong, 2012).
The biochemical assay shows that the kinetics of Crag GEF activity is slow when compared to the GEF activity of other DENN-domain-containing proteins such as the Rab35 GEF. Crag exhibits GEF activity against Rab10 with much faster kinetics than against Rab11, indicating that the slow kinetics may be due to properties of Rab11. This is further supported by the slow kinetics of EDTA that triggers GDP release of Rab11. It's possible that the GDP/GTP exchange of Rab11 requires other co-factors besides its GEF, as, for example, documented for Rab6 (Xiong, 2012).
CaM is a ubiquitously expressed calcium sensor. In the Drosophila photoreceptor cells, photoactivation leads to influx of Ca2+ and activation of CaM. It has been shown that CaM is required for the termination of the photoresponse in several steps, including TRP inactivation and conformational change of metaRh. Crag contains a CaM binding site and interacts with CaM in a calcium-dependent manner. In an in vitro GEF assay, the presence of CaM and Ca2+ indeed enhances the GEF activity of Crag. Hence, it is possible that a light-induced increase of intracellular Ca2+ level enhances Crag activity via CaM binding. The activation of Crag/Rab11 then may serve to replenish rhabdomeric Rh1, whose loss is also induced by light stimulation (Xiong, 2012).
In vertebrate rod cells, polarized transport of Rh is mediated by post-Golgi vesicles that bud from the TGN and fuse with the base of the outer segment. Rab11 has been detected on rhodopsin-bearing post-Golgi vesicles in photoreceptors; however, it has not yet been shown that Rab11 is required for Rh trafficking. DENND4 proteins are highly similar to Crag. This study showed that expression of the UAS–human DENND4A construct not only rescues the lethality but also rescues the light-induced photoreceptor degeneration caused by loss of Crag, showing that the molecular function of DENND4A is also conserved. Moreover, three different subtypes of Usher syndrome, an inherited condition characterized by hearing loss and progressive vision loss, have been mapped to the vicinity of the DENND4A locus at 15q22.31. Hence, DENND4A may also function through Rab11 in human photoreceptors, and loss of DENND4A may lead to photoreceptor degeneration (Xiong, 2012).
date revised: 15 May 2005
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