InteractiveFly: GeneBrief

Rab6: Biological Overview | References

BIOLOGICAL OVERVIEW

The Drosophila body axes are defined by the precise localization and the restriction of molecular determinants in the oocyte. Polarization of the oocyte during oogenesis is vital for this process. The directed traffic of membranes and proteins is a crucial component of polarity establishment in various cell types and organisms. This study investigated the role of the small GTPase Rab6 in the organization of the egg chamber and in asymmetric determinant localization during oogenesis. Exocytosis is affected in rab6-null egg chambers, which display a loss of nurse cell plasma membranes. Rab6 is also required for the polarization of the oocyte microtubule cytoskeleton and for the posterior localization of oskar mRNA. In vivo, Rab6 is found in a complex with Bicaudal-D, and Rab6 and Bicaudal-D cooperate in oskar mRNA localization. Thus, during Drosophila oogenesis, Rab6-dependent membrane trafficking is doubly required; first, for the general organization and growth of the egg chamber, and second, more specifically, for the polarization of the microtubule cytoskeleton and localization of oskar mRNA. These findings highlight the central role of vesicular trafficking in the establishment of polarity and in determinant localization in Drosophila (Coutelis, 2007).

During polarized exocytosis, secretory vesicles emerging from the TGN are targeted via molecular motors and cytoskeletal tracks to the plasma membrane, where they are tethered. Subsequently, their fusion with the plasma membrane permits the secretion of the vesicle contents, as well as the incorporation of vesicular lipids and proteins into the plasma membrane, allowing membrane growth and the establishment of specific domains. The exocyst complex plays a crucial role in the incorporation of particular membranes and membrane proteins at specific sites or in active domains of the plasma membrane. Consistent with this, Drosophila sec5 mutant egg chambers display mislocalization of other exocyst components, cytoplasmic clusters of actin and a loss of plasma membranes. Thus, Sec5 protein is at the core of the exocyst complex in Drosophila, as is the case in yeast and in mammals (Coutelis, 2007).

Both sec5 null (sec5^E10) and strongly affected rab6^D23D egg chambers display actin and general organization defects, and arrest development during early oogenesis. Similarly, sec5 hypomorphic (sec5^E13) and rab6^D23D egg chambers that develop past stage 7 display phenotypes ranging from wild type to a loss of nurse cell cortical actin and the concomitant presence of ring canal clusters in the nurse cell cytoplasm. The striking parallel between the rab6 and sec5 phenotypes, together with the finding that a loss of Rab6 affects Sec5 localization, suggests that the varying degrees of membrane loss observed in rab6^D23D egg chambers reflects the relative reduction of exocyst-complex function in the egg chamber. Thus, during Drosophila oogenesis, Rab6 promotes Sec5 localization and therefore appears to be important for exocyst-complex organization and function. However, consequent to loss of rab6 function, a striking difference was observed between nurse cells and oocyte in the severity of plasma membrane collapse and Sec5 mislocalization. It is hypothesized that the oocyte acts as a major source of membrane in rab6^D23D egg chambers and/or that multiple exocytic pathways cooperate within the germline cyst to promote cyst development (Coutelis, 2007).

Differences in membrane content between the oocyte and the nurse cells, as well as between the individual nurse cells, are observed as early as the germarium stage in wild-type egg chambers. The fusome, a membranous Spectrin-rich structure derived from the spectrosome, which itself is a precursor organelle present in the germline stem cells, grows asymmetrically through the ring canals during the divisions of the germline cyst, linking each cystocyte. It is thought that the oocyte is the four-ring-canal cell that retains the greater part of fusome during the first division. Furthermore, a Drosophila Balbiani body has recently been discovered, which, together with the fusome, organizes the specific enrichment of organelles in the oocyte throughout oogenesis. It is therefore possible that, in rab6 clones, in which the fusome appears normal, such a mechanism of enrichment of organelles in the oocyte concomitantly ensures that the concentration in the oocyte of any perduring Rab6 protein, thus privileging the growth of the plasma membrane of the oocyte over that of the nurse cells. Supporting this notion is the observation that GFP-tagged Rab6 expressed in the germline is enriched in the oocyte from the early stages of oogenesis (germarium region 2) onwards. Together, the combined actions of a residual Rab6-dependent and of additional Rab6-independent pathways might also permit most rab6^D23D oocytes to maintain sufficient vesicular trafficking to develop past stage 7 (Coutelis, 2007).

The stereotypic organization of affected rab6^D23D egg chambers at mid-oogenesis is striking. The oocyte is connected to open syncytia via its four ring canals, suggesting that the membranes linking nurse cells and oocyte are the most resistant. Furthermore, the growth of the remaining membranes indicates that additional vesicular material is delivered and incorporated into these plasma membranes. This suggests that, in these rab6^D23D egg chambers, sustained vesicle trafficking in the oocyte causes new membrane addition to the oocyte plasma membrane. It is hypothesized that, due to the continuity of the plasma membrane defining the cyst, the oocyte acts as a source of membrane that spreads by lateral diffusion throughout the plasma membrane of the cyst, allowing its growth (Coutelis, 2007).

It appears that Rab6-independent exocytic pathways also contribute to the delivery of vesicular material to the plasma membrane in the Drosophila egg chamber. Indeed, Syx1A is detected on the remaining plasma membrane of both rab6-null and sec5 egg chambers, supporting the existence of a Rab6- and Sec5-independent exocytic pathway mediating protein export. This selective loss of Sec5 from nurse cell membranes in rab6 open syncytia, together with the known functions of the exocyst, suggest a simple explanation for the defects caused by a lack of Rab6 function in oogenesis. It is hypothesized that Rab6-dependent and -independent pathways might differ qualitatively in the proteins whose traffic they mediate, or quantitatively in their relative contributions to the delivery of the same cargo between nurse cells and oocyte. These differences may account for the observed differential requirement for Rab6 in the localization of Sec5 in nurse cell, versus oocyte, plasma membranes (Coutelis, 2007).

Our analysis has revealed two separate functions of Rab6: one is a general role in the organization and growth of the egg chamber, and the other is its specialized role in MT cytoskeleton polarization and oskar mRNA localization. This second function appears specific to Rab6 because, in sec5 mutant egg chambers, Staufen localization is normal and the MT cytoskeleton is correctly organized. Only oskar mRNA, and not Oskar protein, is ectopically detected in rab6^D23D egg chambers. This suggests an impairment of oskar mRNA localization, rather than a defect in its anchoring, in which case Oskar protein would be detected with the detached RNA. Defects in oskar mRNA localization, which relies on MT polarity, could be due to a failure in the focusing of the MT cytoskeleton that is observed in rab6 egg chambers (Coutelis, 2007).

In Drosophila and mammalian cells, BicD is known to regulate MT organization. At mid-oogenesis, Rab6 and BicD cooperation could direct MT organization and/or promote the vesicular transport necessary for oocyte polarization and oskar mRNA localization. Given the implication of membrane trafficking in the asymmetric localization of mRNAs, it also possible that polarized membrane transport along the oocyte MT network directs oskar mRNA to the posterior of the oocyte, by hitch-hiking along trafficking vesicles (Coutelis, 2007).

In MDCK cells, definition of apical and basolateral plasma membrane domains is required during polarization for the arrangement of MT along an apical-basal axis. Vesicular trafficking is crucial to establish, specify and maintain these membrane domains. By analogy, at stage 7, the polarizing signal from the posterior follicular cells to the Drosophila oocyte that causes repolarization of the MT cytoskeleton might do so by inducing the definition of anterior-lateral and posterior membrane domains. It is therefore possible that, in rab6^D23D oocytes, as in epithelia, defects in vesicular trafficking and TGN sorting underlie the observed defects in MT-network organization. Consistent with this idea, a mispolarized MT cytoskeleton is also observed in oocytes lacking Rab11. Thus, vesicular trafficking and the specification of membrane domains may be required for repolarization of the MT network and for the localization of molecular determinants in the Drosophila oocyte at mid-oogenesis (Coutelis, 2007).

Rab6 and the secretory pathway affect oocyte polarity in Drosophila

The Drosophila oocyte is a highly polarized cell. Secretion occurs towards restricted neighboring cells and asymmetric transport controls the localization of several mRNAs to distinct cortical compartments. This study describes a role for the Drosophila ortholog of the Rab6 GTPase, Drab6, in establishing cell polarity during oogenesis. Drab6 localizes to Golgi and Golgi-derived membranes and interacts with BicD. Evidence is provided that Drab6 and BicD function together to ensure the correct delivery of secretory pathway components, such as the TGFα homolog Gurken, to the plasma membrane. Moreover, in the absence of Drab6, osk mRNA localization and the organization of microtubule plus-ends at the posterior of the oocyte were both severely affected. These results point to a possible connection between Rab protein-mediated secretion, organization of the cytoskeleton and mRNA transport (Januschke, 2006).

In vertebrate cells, Rab6 is associated with the Golgi and the trans-Golgi network (TGN) membranes (Del Nery, 2006; Mallard, 2002; Martinez, 1997; Martinez, 1994; Opdam, 2000). To investigate the subcellular localization of Drab6 in the Drosophila germ line, the expression pattern was monitored of transgenic lines expressing Drab6 fused to GFP and RFP. It was observed that during oogenesis, the global distribution of Drab6 evolved. Drab6 first accumulated transiently in a central position during stages 7/8, then is uniformly distributed at the beginning of stage 9 to end up juxtaposed to the entire oocyte cortex. It is noteworthy that promoters of different strengths give similar expression patterns. In addition, the genomic null allele rab6^D23D (Purcell, 1999) is fully rescued by the different lines expressing Drab6 (Januschke, 2006).

Drab6 does not colocalize extensively with ER membranes (labeled with PDI-GFP) (Bobinnec, 2003). Instead, it seems to be differentially associated with two types of Golgi units as revealed by its association with Lava Lamp (Lva and GalT). Lva, a cis-Golgi marker (Papoulas, 2005), colocalizes with Drab6, mainly at the cortex of the oocyte and in nurse cells. A GFP trap protein corresponding to a UDP-galactose:beta-N-acetylglucosamine beta-1,3-galactosyltransferase (GalT) (Morin, 2001), enriches predominantly in Golgi membranes, exhibits a distribution similar to that of GFP-Drab6: it accumulates in the center of the oocyte at stage 8, where it colocalizes with Drab6, and is later confined to the cortex. Importantly, the distribution of Lva and GalT is similar in both matαtubGFP-Drab6, ubiRFP-Drab6 and control oocytes. Given that Lva and GalT markers are not present in the Golgi cisternae that are evenly distributed throughout the oocyte, as documented by electron microscopy (EM) analysis, they might be the hallmark of distinct functional Golgi units, with Drab6 being able to interact with both types of Golgi. Unlike Lva, the distribution of which is only mildly affected, GalT and acetylglucosamine-modified proteins [detected by the wheat germ agglutinin lectin (WGA)] expressed by Golgi structures are abnormally distributed in Drab6 mutants. Moreover, ultrastructural analysis by EM revealed that the ER is abnormally swollen in Drab6 mutant oocytes, and that the Golgi mini-stacks are markedly curved, with partially inflated cisternae (Januschke, 2006).

These morphological effects led to an investigation of the role of Drab6 in the secretory pathway. The polarized secretion of the TGFα-like growth factor Grk was monitored. Grk secretion is restricted to the anterodorsal corner through a rapid transit from the ER towards the Golgi apparatus. In GFP-Drab6-rescued egg chambers, Grk and Drab6 colocalize. In Drab6 mutant oocytes, grk mRNA localization is the same as in wild type. Grk protein, however, is slightly more abundant than in controls and an important fraction extends ventrally. Polarized secretion of Grk lead to the formation of two dorsal appendages on the egg shell. In the absence of Drab6, mislocalized Grk induces ventralization, instead of a dorsalization (multiple dorsal appendages on the egg shell) as observed when Grk is ectopically secreted. Hence, this argues for a specific failure of Grk delivery to the plasma membrane. This phenotype is specific to Drab6 because it could be fully rescued by the GFP-Drab6 transgene (Januschke, 2006).

Next, the intracellular localization of Grk was examined in the absence of Drab6. Grk accumulated frequently in large ring-like particles in the Drab6 mutant, but not in control oocytes. These Grk 'rings', similar to those of yolk granules, did not contain Lva, suggesting that Grk is not blocked in the Golgi. Grk actually accumulates in Drab6 mutants on vesicles stained by LysoTracker, which labels either lysosomes or late endosomes containing yolk granules. Hence, two independent approaches suggest that Grk is not blocked in the Golgi, but is mislocalized to post-Golgi compartments, probably endosomes (Januschke, 2006).

Interestingly, the secretory impairment was also confirmed by Lycopersicon esculentum tomato lectin (LE) detecting modified proteins in the Golgi. In the absence of Drab6, LE revealed abnormal vesicular structures in the oocyte and nurse cells that fail to reach the cortex. EM analysis also demonstrated rupture of the plasma membrane between neighboring nurse cells. Finally, it was observed that GFP-Drab6-rescued egg chambers exhibit an accumulative enrichment of Drab6 at the plasma membrane during oogenesis, which is particularly evident in nurse cells. This is consistent with the involvement of Drab6 in secretion towards the plasmalemma (Januschke, 2006).

The existence of three important and novel aspects of Drab6 function during oogenesis has been established as follows: (1) Consistent with its localization in vertebrate cells, Drab6 is predominantly localized to the Golgi complex in Drosophila, but overlaps with Golgi markers that have distinct localizations, suggesting that Drab6 might associate with distinct functional Golgi units. Drab6 might also play a role in membrane exchange between Golgi and ER and in Golgi organization, according to EM analysis, which is again consistent with known functions of mammalian Rab6 (Del Nery, 2006; Martinez, 1997; Young, 2005). (2) By controlling the migration of Golgi units towards the cell cortex, Drab6 controls the delivery of membrane to the plasmalemma, as shown in Drab6 mutants in which glycosylated proteins labeled by WGA and LE lectins accumulate in large vesicular structures. This pattern is similar to the mislocalization profile of Grk in the absence of Drab6. (3) In the oocyte, Drab6 is required for the anterodorsal secretion of Grk, which leads to the differentiation of the follicle cells required for the morphogenesis of the dorsal appendages of the egg shell. In the absence of Drab6, it was observed that Grk is mislocalized to late endosomal or lysosomal compartments, demonstrating that Drab6 also affects post-Golgi traffic. In vertebrates, one of the Rab6 isoforms (Rab6A') is also involved in endosome-to-Golgi transport (Del Nery, 2006; Utskarpen, 2006). Additionally, a role for Ypt6p (the only copy of Rab6 in the yeast S. cerevisiae) has also been documented as being involved in fusion of endosome-derived vesicles with the late Golgi (Siniossoglou, 2001). It remains to be established whether Drab6 functions directly in the secretory pathway or if the effects observed in Drab6 mutants on post-Golgi trafficking are a consequence of defects in endosome-to-Golgi trafficking (Januschke, 2006).

In order to identify potential Drab6-binding proteins, a yeast two-hybrid screen was performed using as bait Drab6Q71L, a GTPase-deficient mutant. Sixty-two distinct truncated clones of BicD, lacking parts of the amino-terminus, interacted with Drab6Q71L. The intersection of all identified fragments defined a minimal interacting domain, mapping to amino acids 699-772 in the coiled-coil motif H4 of BicD, shown for murine BicD to interact with the mammalian Rab6 (Matanis, 2002). In order to validate this interaction, glutathione S-transferase (GST) pull-down assays were performed, using lysates from wild-type ovaries. GST-Drab6 specifically retained BicD; GST alone and GST-Rab1 did not bind BicD. Furthermore, preloading GST-Drab6 with the non-hydrolyzable GTP analog, GTP-γ-S, yielded an improved interaction with BicD. It is concluded, therefore, that in vitro, BicD interacts through its carboxy-terminus preferentially with the active form of Drab6 (GTP-bound), as has been shown for mammalian Rab6 (Matanis, 2002; Short, 2002; Januschke, 2006 and references therein).

Time-lapse recording showed that in the oocyte and nurse cells, RFP-Drab6 and BicD-GFP colocalize to multiple large aggregates with low dynamics. Further GFP-Drab6 accumulation in the center depends on the presence of BicD during stage 8, as observed in a BicD^mom background. Interestingly, in such BicD^mom oocytes, Grk is found in ring-like structures remote from the nucleus, as observed in Drab6 mutant oocytes (Januschke, 2006).

Since BicD and Rab6 have been shown to be involved in MT-based transport, experiments were conducted to discover whether Drab6-positive structures require MTs for movement. Time-lapse microscopy revealed that large aggregates are less dynamic than the highly motile small particles. Colchicine MT depolymerization severely reduced the movement of Drab6 particles, which form large clusters, indicating that Drab6 is actively transported along MTs. The MT motors Kinesin I [Kinesin heavy chain (Khc)] and Dynein have been shown to be involved in polarizing the Drosophila oocyte. Inactivating the Dynein complex by the overexpression of Dynamitin (Januschke, 2002) prevents accumulation of Drab6 at the oocyte cortex, but does not significantly reduce Drab6 movements. By contrast, in Khc^7.288 germ line clones, Drab6 does not localize in the center of the oocyte during stage 7/8 but forms abnormal aggregates around the mispositioned nucleus. For reasons not currently fully understood, the speed of Drab6 particles is significantly reduced compared with controls or Dynamitin-overexpressing oocytes (Januschke, 2006).

Drab6 and BicD interact in a yeast two-hybrid screen and in GST pull-down assays and colocalize in vivo. Moreover, there are indications that Drab6 requires BicD for correct subcellular localization, which suggests that Drab6 interacts with BicD in Drosophila as it does in mammals. Strikingly, it was found that lack of each protein compromises Grk secretion in a very similar way. Overexpression of Dynamitin, to impair Dynein function, induces ectopic accumulation of Grk and ventralization of the egg shell (Januschke, 2002). Therefore, in Drosophila, BicD/Dynein and Drab6 are likely to be involved together in Grk secretion to the anterodorsal corner of the oocyte (Januschke, 2006).

It is important to mention that colocalization of the two proteins is limited. Moreover, lack of BicD or Drab6 yields different phenotypes. BicD mutation affects oocyte determination and the position of the oocyte nucleus, but has no impact on MT organization in mid-oogenesis, which is not the case in the Drab6 mutant. A genetic interaction between BicD's co-factor Egalitarian and Kinesin I has already been demonstrated, suggesting that Drab6 might interact with Dynein and Kinesin I via BicD (Januschke, 2006).

Interestingly, it was noticed that in the absence of Drab6, osk mRNA is not correctly localized in the oocyte. gurken and bicoid mRNAs are, however, unaffected, and osk mRNA localization to the oocyte center is frequent when the MT network is not correctly polarized. In Drab6 mutant oocytes, the defective posterior localization of the MT plusend marker Khc-ß-Gal indicates a defect in MT organization (Januschke, 2006).

Given that Drab6 is required for late Grk signaling at the anterodorsal corner of the oocyte, it might also be involved in early germ line to soma signaling mediated by Grk, which controls MT organization. This is thought unlikely. In the absence of this signaling, posterior follicle cells differentiate into anterior follicle cells and, as a consequence, the posterior structure of the egg shell, the aeropyle, is substituted with an anterior structure, the micropyle. An aeropyle is always observed at the posterior of eggs derived from Drab6 mutant oocytes. Additionally, removing Drab6 from the posterior follicle cells does not affect oocyte polarity. Hence, Drab6 is possibly involved in MT organization at the posterior pole. Interestingly, Rab6 family interactors such as Rab6IP2/ELKS (Monier, 2002) are capable of interacting with CLASPs at the cortex of HeLa cells (Lansbergen, 2006), suggesting a link between Rab6 protein and MT organization at the cortex (Januschke, 2006).

Toward a comprehensive map of the effectors of Rab GTPases

The Rab GTPases recruit peripheral membrane proteins to intracellular organelles. These Rab effectors typically mediate the motility of organelles and vesicles and contribute to the specificity of membrane traffic. However, for many Rabs, few, if any, effectors have been identified; hence, their role remains unclear. To identify Rab effectors, a comprehensive set of Drosophila Rabs was used for affinity chromatography followed by mass spectrometry to identify the proteins bound to each Rab. For many Rabs, this revealed specific interactions with Drosophila orthologs of known effectors. In addition, numerous Rab-specific interactions with known components of membrane traffic as well as with diverse proteins not previously linked to organelles or having no known function. Over 25 interactions were confirmed for Rab2, Rab4, Rab5, Rab6, Rab7, Rab9, Rab18, Rab19, Rab30, and Rab39. These include tethering complexes, coiled-coiled proteins, motor linkers, Rab regulators, and several proteins linked to human disease (Gillingham, 2014).

The dimeric Golgi protein Gorab binds to Sas6 as a monomer to mediate centriole duplication

Gorab shows many of the properties typical of golgins, a family of tentacle-like proteins that protrude from the Golgi membranes to capture a variety of target vesicles. Redundancy between golgins in their ability to bind target vesicles could act as a functional safeguard and might explain why loss-of-function gorab mutants display no obvious Golgi phenotype, contrasting to the Golgi defects shown by the C-terminally tagged Gorab molecule (Kovacs, 2018). Gorab is similar to other golgins, which also associate with the Golgi membranes through their C-terminal parts in interactions that require Rab family member proteins to interact with the C-terminal part of the golgin dimer. The N-terminal parts of the golgins interact with their vesicle targets. Human GORAB's N-terminal part interacts with Scyl1 to promote the formation of COPI vesicles at the trans-Golgi (Witkos, 2019). However, its precise role in the transport of COPI vesicles is not clear, particularly why loss of human GORAB affects Golgi functions in just bone and skin when COPI function is required in multiple tissues. Drosophila Gorab also co-purifies and physically interacts with both Yata, counterpart of Scyl1, and COPI vesicle components, and its importance for transport of COPI vesicles in Drosophila is similarly unclear (Fatalska, 2021).

This study offers a perspective on how Gorab interacts with Sas6 at the centriole and suggests the possibilities for why this interaction is essential to establish the centriole's ninefold symmetry. The heterotrimeric structure formed by a Sas6 dimer and the Gorab monomer will together constitute a single spoke plus central hub unit of the centriole's cartwheel. The C-terminal part of Gorab would be expected to lie in a tight antiparallel association with the C-terminal part of Sas6's coiled-coil region. Gorab's N-terminus might thus be expected to extend towards the centriolar microtubules and their associated proteins. As the microtubules of Drosophila's somatic centrioles exist as doublets of A- and B-tubules, it is tempting to speculate that Gorab interacts with the centriole wall in a region occupied in other cell types by the C-tubule. This could account for the lack of any requirement for Sas6-Gorab interaction in the male germ-line, where centrioles have triplet microtubules and a C-tubule occupies this space. Gorab's partner proteins interacting with its N-terminal region are therefore of great interest at both the Golgi and in the centriole, and it will be key to understand the nature of these interactions in future studies (Fatalska, 2021).

Warthog and trafficking of Notch

The warthog (wrt) gene, recovered as a modifier for Notch signaling, was found to encode the Drosophila homolog of rab6, Drab6. This study implicates Warthog in the trafficking of Notch. Translation of the Drab6 sequence shows it to have 89% identity to human rab6, 72% to the yeast rhy1 protein, and it has subsequently been cloned as Drab6. Additionally, two putative C. elegans proteins were also found to be homologous (84% and 75%). Vertebrate and yeast homologs of this protein have been shown to regulate Golgi network to TGN trafficking. RAB proteins comprise the largest class of the ras-like GTPase superfamily. Genetic and biochemical studies have shown their involvement in various steps of endocytosis, exocytosis, and transcytosis. Particular rabs are localized to distinct intracellular compartments, and mutant forms of these proteins impair the trafficking of vesicles from one intracellular compartment to another. Rabs have largely been implicated in the fusion or docking of vesicles to acceptor compartments, although some reports have noted rab function in the budding of vesicles from the donor compartment. As GTPases, they act as cyclical switches, alternating between an active GTP-bound state and an inactive GDP-bound state. RabGDI extracts the GDP-bound form from membranes of the acceptor compartment and maintains the rab in this inactive state in the cytosol. Guanine nucleotide exchange factors then promote the exchange of GDP to GTP, converting the rab to an active state, which is presumed to bind to membranes of the donor compartment. Once bound to GTP, hydrolysis of the nucleotide occurs constitutively, providing a timer for the length of rab activation. To slow this constitutive hydrolysis, effector proteins bind to the GTP-bound rab, providing extended time for the complex to target the donor vesicle to the appropriate acceptor compartment. Rab then proceeds through the cycle again. One of these proteins, rab6, has been shown to regulate trafficking from the Golgi to the TGN. In mammalian tissue culture cells, mutations in rab6 lead to morphological changes in the Golgi and a delay in the presentation of proteins to the cell surface. In yeast, null mutations of the rab6 homologues, Ypt6 and rhy1, also show defects in post-ER processing of various proteins. Sequences homologous to rab 6 have also been found in Drosophila, but only structural data have been reported (Purcell, 1999).

To study the function of Drab6 protein in the development of a multicellular organism, three different warthog mutants of Drosophila were analyzed. The first was an R62C point mutation, the second a genomic null, and the third was an engineered GTP-bound form. Contrary to yeast, the Drosophila homologue of rab6 is an essential gene. However, it has limited effects on development beyond the larval stage. Only the mechanosensory bristles on the head, notum, and scutellum are affected by warthog mutations. warthog, enhances the Notch eye phenotype although it does not visibly affect eye development outside of this interaction. It was also noted to have a recessive bristle phenotype independent of its interaction with the aberrant Notch signaling in the eye. In wild-type flies, bristles are part of mechanosensory organs and develop shortly after puparium formation as the trichogen, or shaft cell, sends a cytoplasmic extension from the epidermis into the overlying cuticulin. At the center of this extension is a longitudinal core of microtubules. Around the circumference and positioned near the plasma membrane are regularly spaced bundles of actin filaments. These filaments are hexagonally packed and run parallel to the microtubule core. As development proceeds, continued growth of the shaft occurs in two directions. One is elongation at the distal tip, whereas the second is throughout the width of the shaft as regions of the cytoplasm protrude from between the actin fibers to produce the characteristic ridges seen in a cross-section of the bristle. Five warthog alleles recovered in the screen had considerably shortened bristles as homozygotes or transheterozygotes. This defect was present only for macrochaete of the ocelli, notum, and scutellum, whereas the bristles of the eye, wing, and leg appeared normal. Scanning electron micrographs of warthog bristles show, in addition to the aberrant length, that the morphology of wrt bristles are altered. The wrt bristles do not have finely tapered ends nor do they show the regularly spaced ridges from the membranous protrusions. Instead, the tips are mangled and the surface is either smooth or has very mild and disorganized ruffling. Unexpectedly, a subset of the Drab6 cDNA transformant lines rescue the lethality to produce flies with bristle defects more subtle than the original wrt alleles. Since these same transformant lines are capable of rescuing the bristle defect of the screen alleles with the R62C point mutation, this indicates that bristle development is more sensitive to the quantity or timing of Drab6 expression or function than is lethality (Purcell, 1999).

To establish the time period of Drab6 expression critical for viability, homozygotic mutants were monitored at different stages of development. Eggs with these homozygotic genotypes would proceed through embryogenesis to the larval stage, but would not continue to develop into pupae. Therefore, the more severe alleles of warthog are larval lethal. To determine if the lack of embryonic lethality is due to a maternal contribution of wrt, the FLP-FRT system was used to generate females with wrt-/wrt- germlines. All progeny germline mutants develop past embryogenesis, showing that a maternal contribution is not responsible for survival of wrtP2352 through embryonic development. To study the effect of the more severe disruptions in Drab6 function during later stages of development, mosaic clones were induced using the FLP-FRT system. As with the original screen mutants, no defects of eye, wing, or leg development were noted. The defects on macrochaete are more severe and more variable than that seen with the homozygous mutants. Mutation clones also affect the smaller bristles, called microchaete, on the head and thorax. The defects seen in these smaller shafts mirror those seen in the macrochaete of mutant flies; distal tip growth is stunted and the circumferential ridges produced from cytoplasmic protrusions are nearly absent. Surprisingly, the clonal analysis also shows that the phenotypic effect is nonautonomous. Whereas portions of the mosaic clones contained mutant bristles, phenotypically wild-type bristles are also present in patches of mutant tissue, indicating that Wrt protein is not required within the cell producing the shaft of the bristle (Purcell, 1999).

The function of rab proteins in mammalian systems has been elicited by studying the effects of overexpression of wild-type and mutant forms of these proteins. The best characterized forms are those modeled after ras mutations and are known to alter the ability of rabs to cycle between the GDP- and the GTP-bound states. The state of continued GTP binding has been produced by altering the Q of the second conserved GTP-binding domain to a leucine (Q72L in mammalian rab6). This abolishes intrinsic GTPase activity and decreases GAP-stimulated hydrolysis as well. To study the effects of this mutation in the whole organism, a similar mutation was induced in warthog (Drab6-Q71L) . cDNAs of wild-type Drab6, the R62C mutation, and the Q72L mutation were placed under the control of the heat shock promoter to drive expression at different stages of development. Whereas overexpression of the wild-type form and the R62C mutation produces no visible phenotype in the background of wrt+/wrt+, the Q71L mutation alters the direction of bristle growth at any point along the bristle shaft. Overexpression of this GTP-bound mutant produces smoothly curving bristles or bristles with sharp changes in the orientation of growth, followed by continued growth in two opposite directions. Normal morphology appeared distal to the alteration, presumably because of the return of normal Drab6 function after the pulsed overexpression of Drab6 Q71L has passed. Aberrations in the circumferential ridges are also seen, indicating that the membranous protrusions from between the actin bundles are also disrupted. Interestingly, basal expression of the Q71L mutant cDNA without heat shock, is capable of rescuing the bristle phenotype of the R62C alleles, indicating that even small amounts of the Q71L form of Drab6 can rescue the phenotypic effects of the loss-of-function R62C mutation (Purcell, 1999).

What is the relationship between Notch and rab6? The Notch signal transduction pathway is used in many species to modulate the ability of precursor cells to respond to developmental cues. This signal is activated by the binding of the ligand Delta to its receptor Notch to activate downstream proteins. However, the selection for which cells undergo this activation is influenced by the amount of the Notch receptor at the cell surface; Notch is one of only a handful of genes to produce a visible phenotype with either an extra copy of the gene or when missing one copy. Mammalian and yeast forms of Rab6 are involved in Golgi trafficking. In mammalian tissue culture cells, a mutation (Q72L) in rab6 that impairs GTP hydrolysis, leads to a morphological disruption of Golgi structures and a decrease of marker proteins in the late Golgi network. Conversely, a mutation resulting in a GDP-bound form of rab6 (T27N) shows more prominent Golgi structures and an accumulation of marker proteins in the late Golgi network. Both of these rab6 mutations lead to a kinetic inhibition of proteins presented to the cell surface; in pulse-chase experiments, cells that overexpress wild-type or either mutant form of rab6 (Q72L or T27N) eventually secrete the same quantity of extracellular proteins as controls, but the rate of release is markedly decreased. From these tissue culture experiments, mutations in Drab6 would be expected to delay the surface presentation of the Notch receptor. Given that the amount of Notch present on the cell surface is critical for the adoption of different cellular identities, such a delay in transportation of the Notch receptor to the plasma membrane would alter Notch signaling. The phenotypic interaction of the wrt screen alleles was consistent with a decrease in the amount of N available for signaling on the cell surface (Purcell, 1999).

Another explanation for the modification of Notch signaling by wrt is suggested by the observation that rab6 specifically functions at the critical junction of sorting between the amyloidogenic and nonamyloidogenic pathways for the ß-amyloid precursor protein. This role of rab6 in the proper sorting of molecules into different compartments within or from the TGN may account for the interaction between Notch and warthog. Notch undergoes proteolytic cleavage by a furin-like convertase within the TGN to produce a heterodimeric receptor at the cell surface. If rab6 determines which Golgi and post-Golgi enzymes transported proteins encounter, then alterations in warthog function could potentially lead to a missorting of Notch into a transport pathway where the receptor is not cleaved properly (Purcell, 1999 and references).

A rab6-interacting protein, rabkinesin-6, has been shown to bind microtubules and has ATPase activity similar to the plus end motors to which it is homologous; rab6-GTP was postulated to regulate the association and dissociation of rabkinesin-6 to microtubules. However, for warthog, no additive or synergistic interactions were seen when tested with many mutations known to affect bristle structure. More importantly, the nonautonomous phenotype seen in the severe warthog mutants implies the Drosophila homolog of rab6 modifies the surface presentation of other proteins. Nonautonomous phenotypes are typically seen with secreted or transmembrane proteins that signal to neighboring cells. This effect is consistent with results from yeast and mammalian tissue culture experiments that establish the role of rab6 in the proper secretion of other proteins (Purcell, 1999 and references).

Mutations that have previously been studied for rab6 are those engineered based on the GTP- and GDP-bound forms of ras-like molecules. From the screen for modifiers of Notch, a novel mutation was obtained that results in the conversion of an arginine to a cysteine at amino acid 62 (R62C). From biochemical and crystallographic data of other GTPases, the R62C mutation is expected to lie next to a defined GTP-binding domain (DX2G) where the invariant aspartate binds the catalytic Mg2+ through an intervening water molecule. However, in vitro studies reveal that R62C mutant protein is capable of binding and hydrolyzing GTP, suggesting that this point mutation affects Drab6 function through another mechanism, perhaps by altering its interaction with regulatory proteins. This hypomorphic mutation altered rab6 functions differently from the Q71L mutation, which resides next to the same GTP-binding domain. Overexpression Q71L Drab6 disrupts the orientation of bristle growth, whereas overexpression of R62C Drab6 in a wild-type background elicits no effects. Q71L Drab6 is also capable of rescuing the bristle defect of the R62C mutation. Therefore, studying the R62C mutation may reveal new information of Drab6 function (Purcell, 1999 and references).

Perhaps the most interesting aspect of this phenotypic analysis is the limited requirement of a rab6 homolog throughout development. While an essential gene, Drab6 mutations do not affect the development of the eye, wing, and leg, nor the bristle structures within these tissues. This paucity of developmental phenotypes mirrors yeast studies that show null mutations in Ypt6 or rhy1 are not lethal, implying transport redundancy exists as proteins travel to the cell membrane. This redundancy could be the result of more than one rab6 protein, which is supported by the discovery of two putative rab6 homologs in C. elegans. Alternatively, it may be a functional redundancy where parallel but independent trafficking pathways through the Golgi/TGN can compensate for alterations in one another. Recent studies in mammalian systems support the existence of these independent trafficking pathways. The secreted protein ß-APP is processed in a different compartment if rab6 is mutated and a study of cell surface antigen presentation has also shown alterations in rab6 affected one transport pathway but not another (Purcell, 1999 and references).

The bristle phenotype of the warthog mutants, however, reveals there is a limitation to which an organism can compensate for mutations in Drab6, even if redundant or independent pathways exist for transport through the Golgi. This limitation may also be seen only after prolonged Drab6 dysfunction. Overexpression of Drab6 Q71L in a subset of cells within the eye leads to degeneration after two weeks. Having phenotypes associated with this limitation in redundancy through the Golgi/TGN will provide a novel means to dissect Golgi transport mechanisms. Identifying proteins that modify the wrt bristle phenotype will allow an ordered dissection of the protein cascade required for rab6 function. These mutants may also lead to a better understanding of how the cell regulates trafficking of signaling receptors such as Notch. Capitalizing on the interaction between wrt and Notch in sensitized backgrounds, genetic screens may help identify the proteins required for surface presentation of a functional Notch receptor (Purcell, 1999 and references).

Rab6 regulation of rhodopsin transport in Drosophila

Rab6 is a GTP binding protein that regulates vesicular trafficking within the Golgi and post-Golgi compartments. Wild-type, a GTPase defective (Q71L), and a guanine nucleotide binding defective (N125I) Rab6 protein were overexpressed in Drosophila photoreceptors to assess the in vivo role of Rab6 in the trafficking of rhodopsin and other proteins. Expression of Drab6^Q71L greatly reduced the steady state levels of two rhodopsins, Rh1 and Rh3, whereas Drab6^wt and Drab6^N125I showed weaker effects. Analysis of a strain carrying Rh1 rhodopsin under a heat shock promoter showed that Drab6^Q71L, but not Drab6^wt or Drab6^N125I, prevents the maturation of rhodopsin beyond an immature 40 kDa form. Drab6^Q71L is a GTPase defective mutant, indicating that anterograde transport of rhodopsin requires Rab6 GTPase function. The three Drab6 strains had no effect on the expression of several other photoreceptor proteins. The Drab6^Q71L photoreceptors show marked histological defects at young ages and degenerate over a two week time span. These results establish that rhodopsin is transported via a Rab6 regulated pathway and that defects in trafficking pathways lead to retinal degeneration (Shetty, 1998; full text of article).

Constitutive exocytosis delivers newly synthesized proteins, lipids, and other molecules from the Golgi apparatus to the cell surface. This process is mediated by vesicles, which bud off the trans-Golgi network, move along cytoskeletal filaments, and fuse with the plasma membrane. The small GTPase Rab6 marks exocytotic vesicles and, together with the microtubule plus-end-directed motor kinesin-1, stimulates their processive microtubule-based transport to the cell periphery. Furthermore, Rab6 directs targeting of secretory vesicles to plasma-membrane sites enriched in the cortical protein ELKS, a known Rab6 binding partner. These data demonstrate that although Rab6 is not essential for secretion, it controls the organization of exocytosis within the cellular space (Grigoriev, 2007).

Taken together, these data show that Rab6 is abundantly present on exocytotic vesicles and is needed to regulate their behavior. The data do not preclude the involvement of Rab6 GTPase in recycling from Golgi to ER; they do indicate, however, that in contrast to previously published studies, the major target for Rab6-vesicle fusion is the plasma membrane and not the ER. These findings help to explain the recently discovered exocytosis defects in Rab6 mutants during Drosophila oogenesis (Coutelis, 2007) as well as the observations on the role of Rab6 in the trafficking of membrane proteins such as rhodopsin (Deretic, 1998). In addition to its role at the Golgi, Rab6 regulates exocytosis by enhancing processive kinesin-dependent motion of secretory vesicles from the Golgi to MT plus ends. Furthermore, Rab6 is required for targeting these vesicles to the cortical ELKS-containing patches where MT plus ends are attached (Lansbergen, 2006). Therefore, although Rab6 is not essential for anterograde transport, it plays an important role in the spatial organization of constitutive exocytosis (Grigoriev, 2007).

Search PubMed for articles about Drosophila Rab6

Bobinnec, Y., Marcaillou, C., Morin, X. and Debec, A. (2003). Dynamics of the endoplasmic reticulum during early development of Drosophila melanogaster. Cell Motil. Cytoskeleton 54: 217-225. PubMed ID: 12589680

Coutelis, J. B. and Ephrussi, A. (2007). Rab6 mediates membrane organization and determinant localization during Drosophila oogenesis. Development 134(7): 1419-30. PubMed ID: 17329360

Del Nery, E., Miserey-Lenkei, S., Falguieres, T., Nizak, C., Johannes, L., Perez, F. and Goud, B. (2006). Rab6A and Rab6A' GTPases play non-overlapping roles in membrane trafficking. Traffic 7: 394-407. PubMed ID: 16536738

Deretic, D. (1998). Post-Golgi trafficking of rhodopsin in retinal photoreceptors. Eye 12: 526-30. PubMed ID: 9775213

Egerer, J., Emmerich, D., Fischer-Zirnsak, B., Chan, W. L., Meierhofer, D., Tuysuz, B., Marschner, K., Sauer, S., Barr, F. A., Mundlos, S. and Kornak, U. (2015). GORAB missense mutations disrupt RAB6 and ARF5 binding and Golgi targeting. J Invest Dermatol 135(10): 2368-2376. PubMed ID: 26000619

Fatalska, A., Stepinac, E., Richter, M., Kovacs, L., Pietras, Z., Puchinger, M., Dong, G., Dadlez, M. and Glover, D. M. (2021). The dimeric Golgi protein Gorab binds to Sas6 as a monomer to mediate centriole duplication. Elife 10. PubMed ID: 33704067

Gillingham, A. K., Sinka, R., Torres, I. L., Lilley, K. S. and Munro, S. (2014). Toward a comprehensive map of the effectors of Rab GTPases. Dev Cell 31: 358-373. PubMed ID: 25453831

Grigoriev, I., et al. (2007). Rab6 regulates transport and targeting of exocytotic carriers. Dev. Cell 13(2): 305-14. PubMed ID: 17681140

Januschke, J., Gervais, L., Dass, S., Kaltschmidt, J. A., Lopez-Schier, H., Johnston, D. S., Brand, A. H., Roth, S. and Guichet, A. (2002). Polar transport in the Drosophila oocyte requires Dynein and Kinesin I cooperation. Curr. Biol. 12: 1971-1981. PubMed ID: 12477385

Januschke, J., Gervais, L., Gillet, L., Keryer, G., Bornens, M. and Guichet, A. (2006). The centrosome-nucleus complex and microtubule organization in the Drosophila oocyte. Development 133: 129-139. PubMed ID: 16319114

Januschke, J., Nicolas, E., Compagnon, J., Formstecher, E., Goud, B. and Guichet, A. (2007). Rab6 and the secretory pathway affect oocyte polarity in Drosophila. Development 134(19): 3419-25. PubMed ID: 17827179

Kovacs, L., Chao-Chu, J., Schneider, S., Gottardo, M., Tzolovsky, G., Dzhindzhev, N. S., Riparbelli, M. G., Callaini, G. and Glover, D. M. (2018). Gorab is a Golgi protein required for structure and duplication of Drosophila centrioles. Nat Genet. PubMed ID: 29892014

Lansbergen, G., Grigoriev, I., Mimori-Kiyosue, Y., Ohtsuka, T., Higa, S., Kitajima, I., Demmers, J., Galjart, N., Houtsmuller, A. B., Grosveld, F., et al. (2006). CLASPs attach microtubule plus ends to the cell cortex through a complex with LL5beta. Dev. Cell 11: 21-32. PubMed ID: 16824950

Mallard, F., Tang, B. L., Galli, T., Tenza, D., Saint-Pol, A., Yue, X., Antony, C., Hong, W., Goud, B. and Johannes, L. (2002). Early/recycling endosomes-to-TGN transport involves two SNARE complexes and a Rab6 isoform. J. Cell Biol. 156: 653-664. PubMed ID: 11839770

Martinez, O., Schmidt, A., Salamero, J., Hoflack, B., Roa, M. and Goud, B. (1994). The small GTP-binding protein rab6 functions in intra-Golgi transport. J. Cell Biol. 127: 1575-1588. PubMed ID: 7798313

Martinez, O., Antony, C., Pehau-Arnaudet, G., Berger, E. G., Salamero, J. and Goud, B. (1997). GTP-bound forms of rab6 induce the redistribution of Golgi proteins into the endoplasmic reticulum. Proc. Natl. Acad. Sci. USA 94: 1828-1833. PubMed ID: 9050864

Matanis, T., Akhmanova, A., Wulf, P., Del Nery, E., Weide, T., Stepanova, T., Galjart, N., Grosveld, F., Goud, B., De Zeeuw, C. I., et al. (2002). Bicaudal-D regulates COPI-independent Golgi-ER transport by recruiting the dynein-dynactin motor complex. Nat. Cell Biol. 4: 986-992. PubMed ID: 12447383

Monier, S., Jollivet, F., Janoueix-Lerosey, I., Johannes, L. and Goud, B. (2002). Characterization of novel Rab6-interacting proteins involved in endosome-to-TGN transport. Traffic 3: 289-297. PubMed ID: 11929610

Morin, X., Daneman, R., Zavortink, M. and Chia, W. (2001). A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila. Proc. Natl. Acad. Sci. USA 98: 15050-15055. PubMed ID: 11742088

Opdam, F. J., Echard, A., Croes, H. J., van den Hurk, J. A., van de Vorstenbosch, R. A., Ginsel, L. A., Goud, B. and Fransen, J. A. (2000). The small GTPase Rab6B, a novel Rab6 subfamily member, is cell-type specifically expressed and localised to the Golgi apparatus. J. Cell Sci. 113: 2725-2735. PubMed ID: 10893188

Papoulas, O., Hays, T. S. and Sisson, J. C. (2005). The golgin Lava lamp mediates dynein-based Golgi movements during Drosophila cellularization. Nat. Cell Biol. 7: 612-618. PubMed ID: 15908943

Purcell, K. and Artavanis-Tsakonasa, S. (1999). The developmental role of warthog, the notch modifier encoding Drab6. J. Cell Biol. 146: 731-740. PubMed ID: 10459009

Shetty, K. M., Kurada, P. and O'Tousa, J. E. (1998). Rab6 regulation of rhodopsin transport in Drosophila. J. Biol. Chem. 273(32): 20425-30. PubMed ID: 9685396

Short, B., et al. (2002). The Rab6 GTPase regulates recruitment of the dynactin complex to Golgi membranes. Curr. Biol. 12(20): 1792-5. PubMed ID: 12401177

Siniossoglou, S. and Pelham, H. R. (2001). An effector of Ypt6p binds the SNARE Tlg1p and mediates selective fusion of vesicles with late Golgi membranes. EMBO J. 20: 5991-5998. PubMed ID: 11689439

Utskarpen, A., Slagsvold, H. H., Iversen, T. G., Walchli, S. and Sandvig, K. (2006). Transport of ricin from endosomes to the Golgi apparatus is regulated by Rab6A and Rab6A'. Traffic 7: 663-672. PubMed ID: 16683916

Witkos, T. M., Chan, W. L., Joensuu, M., Rhiel, M., Pallister, E., Thomas-Oates, J., Mould, A. P., Mironov, A. A., Biot, C., Guerardel, Y., Morelle, W., Ungar, D., Wieland, F. T., Jokitalo, E., Tassabehji, M., Kornak, U. and Lowe, M. (2019). GORAB scaffolds COPI at the trans-Golgi for efficient enzyme recycling and correct protein glycosylation. Nat Commun 10(1): 127. PubMed ID: 30631079

Young, J., Stauber, T., del Nery, E., Vernos, I., Pepperkok, R. and Nilsson, T. (2005). Regulation of microtubule-dependent recycling at the trans-Golgi network by Rab6A and Rab6A'. Mol. Biol. Cell 16: 162-177. PubMed ID: 15483056

date revised: 5 December 2023

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