InteractiveFly: GeneBrief

Rab1: Biological Overview | References

Gene name - Rab1

Synonyms -

Cytological map position - 93D2-93D2

Function - signaling

Keywords - controls membrane trafficking and contractile ring constriction during cytokinesis, regulates ER-to-Golgi transport, essential for dendrite pruning of ddaC neurons, promotes endocytosis and downregulation of the cell-adhesion molecule Neuroglian and thereby dendrite pruning, controls material delivery from Golgi to the plasma membrane

Symbol - Rab1

FlyBase ID: FBgn0285937

Genetic map position - chr3R:21,266,029-21,269,060

NCBI classification - Ras family, NTPase: P-loop containing Nucleoside Triphosphate Hydrolases

Cellular location - cytoplasmic

NCBI links: EntrezGene, Nucleotide, Protein

Cytokinesis requires a tight coordination between actomyosin ring constriction and new membrane addition along the ingressing cleavage furrow. However, the molecular mechanisms underlying vesicle trafficking to the equatorial site and how this process is coupled with the dynamics of the contractile apparatus are poorly defined. This study provides evidence for the requirement of Rab1 during cleavage furrow ingression in cytokinesis. The gene omelette (omt) encodes the Drosophila orthologue of human Rab1 and is required for successful cytokinesis in both mitotic and meiotic dividing cells of Drosophila melanogaster. Rab1 protein was shown to colocalizes with the conserved oligomeric Golgi (COG) complex Cog7 subunit and the phosphatidylinositol 4-phosphate effector GOLPH3 at the Golgi stacks. Analysis by transmission electron microscopy and 3D-SIM super-resolution microscopy reveals loss of normal Golgi architecture in omt mutant spermatocytes indicating a role for Rab1 in Golgi formation. In dividing cells, Rab1 enables stabilization and contraction of actomyosin rings. It was further demonstrated that GTP-bound Rab1 directly interacts with GOLPH3 and controls its localization at the Golgi and at the cleavage site. It is proposed that Rab1, by associating with GOLPH3, controls membrane trafficking and contractile ring constriction during cytokinesis (Sechi, 2017).

Cytokinesis represents the final act of cell division when a mother cell becomes fully partitioned into two daughter cells. Cytokinesis failures can contribute to several human diseases including blood disorders, age-related macular degeneration, Lowe syndrome, female infertility and cancer. In animal cells, cytokinesis relies upon constriction of a plasma membrane-anchored actomyosin ring, which leads to cleavage furrow ingression at the equatorial cortex. To fully separate each mother cell into two daughter cells, cytokinesis is also associated with a considerable expansion of cell plasma membrane. Insertion of new membrane during cytokinesis is achieved through shuttling of membrane vesicles to the ingressing cleavage furrow and involves both secretory and endocytic/recycling trafficking activities. Accumulating evidence also indicates that phosphoinositide lipids regulate both contractile ring dynamics and membrane trafficking during cytokinesis. Drosophila male meiosis provides an excellent cell system to dissect the vesicle trafficking pathways involved in cytokinesis. Indeed screens for mutants affecting spermatocyte cytokinesis have identified several components of the Golgi and endocytic/recycling machinery, comprising the conserved oligomeric Golgi complex (COG) subunits Cog5 and Cog7, the TRAPPII complex subunit Brunelleschi, the Dyntaxin 5 ER-to-Golgi vesicle-docking protein, the small GTPases Rab11 and Arf6, the COPI subunits and the exocyst complex proteins Sec8 and Exo84. Mutations affecting male meiotic cytokinesis have also revealed the requirement for proteins that regulate the phosphoinositide pathway including the Drosophila Phosphatidylinositol (PI) transfer protein (PITP), Giotto/Vibrator (Gio/Vib) and the PI 4-kinase III β Four wheel drive (Fwd). Both Fwd and Gio/Vib are required to localize Rab11 at the cleavage site. Fwd directly binds Rab11 at the Golgi and is required for synthesis of PI 4-phosphate (PI(4)P) on Golgi membranes and for localization of secretory organelles containing both PI(4)P and Rab11 at the cleavage site. Recent work has shown that the oncoprotein GOLPH3, described as a PI(4)P effector at the Golgi (Sechi, 2015a & b), accumulates at the cell equator of dividing cells and is required for cleavage furrow ingression in Drosophila (Sechi, 2014). GOLPH3 function during cytokinesis is intimately connected to its ability to bind PI(4)P and regulates both the dynamics of the actomyosin ring and vesicle trafficking to the cleavage site (Sechi, 2017 and references therein).

This study provides the first comprehensive demonstration for Rab1 function in cytokinesis, in tissues of a multicellular organism. A possible involvement of Rab1 in mitotic cytokinesis was previously suggested by a genome-wide screen aimed at identifying genes required for cytokinesis in cultured Drosophila cells, reporting a slight increase of binucleate cells in RNAi-treated cells when compared with control. The current analysis reveals defects in early stages of cytokinesis of dividing spermatocytes, neuroblasts and S2 cells with reduced Rab1 protein expression, which result in incomplete contractile ring constriction. Although myosin II/anillin rings were observed in early telophases of omt/Df mutants, these structures failed to undergo full constriction during cytokinesis. A similar phenotype was found also in Drosophila S2 cells depleted of Rab1. Failure to assemble functional actomyosin rings is a commonly observed phenotype in male meiotic mutants of membrane-trafficking components including the COG subunits Cog5 and Cog7, the Arf6 and Rab11GTPases, the TRAPPII subunit Brunelleschi (Robinett, 2009) and GOLPH3. A model has been suggested whereby, during cytokinesis, assembly and dynamics of the contractile apparatus are intimately connected with vesicle trafficking and membrane remodelling at the cleavage furrow. In this context, membrane vesicles that fuse with the furrow membrane during cytokinesis might also transport structural components of the contractile ring or F-actin regulators. Indeed, visualization of actin and endocytic vesicles in cellularizing Drosophila embryos has suggested that F-actin and vesicles might be targeted as a unit to the furrow site (Sechi, 2017).

The finding that Rab1 localizes at the Golgi suggests a role for this protein in Golgi trafficking during cytokinesis. In agreement with this hypothesis, this analysis of Golgi in interphase spermatocytes by 3D-SIM super-resolution microscopy and TEM revealed a highly altered structure in Rab1 mutants, suggesting that defective trafficking through the Golgi may impair the flow of vesicle trafficking to the cleavage site and halt cytokinesis. Moreover, the characteristic organization of Drosophila Golgi into multiple discrete stacks, scattered throughout the cytoplasm, allowed has led to the uncovering of structural defects, caused by Rab1 mutations, that might not be identified in mammalian cells where the stacks are interconnected into a single ribbon-like Golgi. Indeed, mutations in Rab1 affected both the number and size of the Golgi stacks and disrupted the ultrastructure of Golgi cisternae. Golgi fragmentation is likely to result from defective COP I-mediated vesicle trafficking, which in turn depends on the GTPase Arf1 and its guanine nucleotide exchange factor GBF1. Consistent with this hypothesis, the current work demonstrates that Rab1 interacts with Garz, the Drosophila orthologue of GBF1, which is essential to recruit this protein to the Golgi. Golgi fragmentation and trafficking defects are also likely to result from decreased localization of the PI(4)P-binding protein GOLPH3. Remarkably, GOLPH3 is a key protein for maintaining Golgi architecture and vesicular release (Dippold, 2009). A recent study has proposed that human Rab1B, in complex with PITPNC1, might control Golgi morphology by regulating Golgi PI(4)P levels and hence indirectly the abundance of the PI(4)P effector GOLPH3. In agreement with this work, the current data indicate that GOLPH3 requires wild-type function of Rab1 for its localization at the Golgi membranes during both interphase and telophase. Moreover, this study provides evidence that GOLPH3 protein directly interacts with Rab1-GTP and requires wild-type function of Rab1 for its recruitment to the cleavage site. Taken together these data suggest that Rab1 protein, by contributing to GOLPH3 recruitment, enables secretory vesicle trafficking and actomyosin constriction during cytokinesis. It cannot be excluded that the loss of Rab1 could have additional effects through other Golgi effectors in addition to GOLPH3 and that the cytokinesis defects might be the indirect consequences of altered secretory or endocytic pathways that are known to be important for cytokinesis. Indeed mutations in Rab1 do not affect Golgi localization of Cog7 but disrupt recruitment of the ArfGEF orthologue Garz, a known Golgi effector of Rab1. Nevertheless, these data indicate GOLPH3 is a major effector of Rab1 in mediating contractile ring constriction and cleavage furrow ingression during cytokinesis (Sechi, 2017).

In mammalian cells, a single molecular TRAPPII complex acts as a GDP-GTP exchange factor for Rab1. This complex appears to have a counterpart in Drosophila melanogaster where Bru, the fly orthologue of the TRAPPII subunit Trs120p, is also required for cleavage furrow ingression during male meiotic cytokinesis. In fission yeast and plant cells, this role appears to require both the TRAPPII and exocyst complexes that associate with vesicles in the cleavage furrows. These data suggest a possible conserved role for Rab1 together with the TRAPPII complex in guiding membrane addition to the cleavage furrow during cytokinesis that in animal cells may be played by a single complex. The investigation of such possibilities will be the topic of future work (Sechi, 2017).

Yif1 associates with Yip1 on Golgi and regulates dendrite pruning in sensory neurons during Drosophila metamorphosis

Pruning that selectively removes unnecessary neurites without causing neuronal death is essential for sculpting the mature nervous system during development. In Drosophila, ddaC sensory neurons specifically prune their larval dendrites with intact axons during metamorphosis. However, the important role of endoplasmic reticulum (ER)-to-Golgi transport in dendrite pruning remains unknown. In a clonal screen, this study has identified Yif1, an uncharacterized Drosophila homolog of Yif1p that is known to be a regulator of ER-to-Golgi transport in yeast. Yif1 is shown to be required for dendrite pruning of ddaC neurons but not for apoptosis of ddaF neurons. The Yif1-binding partner Yip1 was shown to be crucial for dendrite pruning. Yif1 forms a protein complex with Yip1 in S2 cells and ddaC neurons. Yip1 and Yif1 colocalize on ER/Golgi and are required for the integrity of Golgi apparatus and outposts. Moreover, two GTPases, Rab1 and Sar1, which are known to regulate ER-to-Golgi transport, are essential for dendrite pruning of ddaC neurons. Finally, the data reveal that ER-to-Golgi transport promotes endocytosis and downregulation of the cell-adhesion molecule Neuroglian and thereby dendrite pruning (Wang, 2018).

The secretory pathway involves three elementary organelles: the endoplasmic reticulum (ER), the Golgi apparatus and the trans-Golgi network (TGN). Via the secretory pathway, protein and lipid supplies are provided to facilitate the specification and outgrowth of dendrites and axons during neuronal development. In hippocampal neurons, the disruption of the secretory pathway through suppressing the post-Golgi trafficking or normal Arf1 function leads to a dendrite-outgrowth defect. Disruption of ER-to-Golgi transport by inhibiting Sar1 activity leads to shortened axons in mammalian neurons. In Drosophila, the disruption of ER-to-Golgi transport by mutating Sar1 or Rab1 dramatically inhibits dendrite arbor elaboration with normal axonal elongation in sensory neurons (Ye, 2007). In addition to facilitating dendrites/axon outgrowth, the secretory pathway is also essential for the maintenance of dendrite arbors after neuron maturation. In stark contrast to its roles in neuronal growth and maintenance, a recent study has reported that Arf1/Sec71-mediated post-Golgi trafficking process is also crucial for dendrite pruning: a regressive event (Wang, 2017). However, a potential role for ER-to-Golgi transport in dendrite pruning remained elusive (Wang, 2018).

This study identified two novel genes, Yif1 and Yip1, that play important roles in dendrite pruning in ddaC sensory neurons during early metamorphosis. Their respective homologs are known to regulate ER-to-Golgi transport in yeast and mammals. Yif1 is shown to associates with Yip1 in S2 cells and ddaC neurons. Moreover, Yip1 and Yif1 colocalize on the ER and cis-Golgi, and both are required for the integrity of the Golgi apparatus and outposts. The small GTPases Rab1 and Sar1, two key regulators of ER-to-Golgi transport, were identified as crucial for dendrite pruning of ddaC neurons. Importantly, the data indicate that the ER-to-Golgi transport promotes dendrite pruning partly via endocytosis and downregulation of the cell-adhesion molecule Neuroglian (Nrg). Thus, these data argue that some yet to be-identified molecules might be secreted into the dendrites to trigger Nrg internalization and dendrite pruning (Wang, 2018).

Drosophila Yif1 and Yip1 belong to the same protein family as yeast Yif1p/Yip1p. Yeast Yip1p was first identified as a Ypt1p/Rab1-interacting protein and Yif1p was further discovered as a Yif1p-interacting factor. Via their C-terminal regions, Yif1p and Yip1p form a heteromeric integral membrane complex. These two proteins localize on the Golgi membranes and regulate ER-to-Golgi protein trafficking and secretion. Yip1p-Yif1p was also reported to regulate the fusion process between ER-derived vesicles and the Golgi apparatus. This complex was proposed to serve as a receptor on the Golgi membranes for vesicle docking and fusion. In mammals, Yif1B was discovered as a binding partner of the 5-HT1A serotonin receptor and localized on intermediate compartments of the Golgi. Partial knockdown of Yif1B specifically disturbs the targeting of the 5-HT1A receptor to the distal dendrites, whereas complete knockout of Yif1B leads to the disruption of Golgi integrity. Likewise, mammalian Yip1 proteins were also discovered as trafficking regulators between ER exit sites, intermediate compartment and cis-Golgi (Wang, 2018).

This study shows that Drosophila Yif1 and Yip form a protein complex in vivo and are functionally relevant during dendrite pruning. First, these two proteins associate in both S2 cells and ddaC sensory neurons, as revealed in co-IP experiments and BiFC assays. Second, they colocalize on ER and cis-Golgi, and importantly their localizations are mutually dependent. Third, both of them are required for the integrity of Golgi apparatus, similar to the COPII-mediated ER-to-Golgi trafficking regulators, such as Rab1 or Sar1. Finally, removal of either of them caused the same phenotypes in terms of dendrite pruning, identical to those observed in Rab1 or Sar1 mutant neurons. Thus, it is plausible to suggest that Drosophila Yif1 and Yip1, like their homologs, can regulate the ER-to-Golgi transport to promote dendrite pruning. Consistent with these findings, previous genome-wide RNAi screens reported Yif1 as a potential component of the secretory pathway that might be involved in neural outgrowth and morphology (Wang, 2018).

Growing evidence indicates that the secretory pathway has been shown to regulate dendrite growth and maintenance in both Drosophila and mammals. The ER-to-Golgi transport is an early step of the canonical secretory pathway. Suppression of the ER-Golgi transport inhibits dendrite outgrowth in the developing neurons. Either disrupting Golgi apparatus or attenuating Golgi outposts alters dendrite morphology and outgrowth in mature neurons. A previous study illustrated that the small GTPas Arf1, which is known to regulate post-Golgi trafficking in mammals, is important for proper dendrite pruning of ddaC sensory neurons (Wang, 2017). Moreover, this present study describes Yif1 and Yip1 as two new regulators of dendrite pruning in sensory neurons. Yif1 and Yip1 appear to localize on ER and cis-Golgi, which is compatible with their roles in the ER-to-Golgi protein transport like their yeast and mammalian homolog. Yif1 and Yip1 can facilitate the biogenesis of secretory vesicles and affect the proper Golgi structure in ddaC neurons. Consistently, Rab1 and Sar1, which are two key components of the COPII vesicles, are also essential for dendrite pruning. Thus, it is tempting to hypothesize that some yet to be-identified molecules might be specifically secreted into the dendrites to promote dendrite pruning. Interestingly, mammalian Yif1B functions as a scaffold protein to recruit the 5-HT1A receptor together with Yip1A and Rab6, probably in the same trafficking vesicles and specifically targets the receptor to the distal dendrites in rat neurons (Al Awabdh, 2012). However, the current results suggest that this machinery might not be required for dendrite pruning of ddaC neurons. First, although the N-terminal 50 amino acid region of mammalian Yif1B is required for targeting the 5-HT1A receptor, this study found that a similar N-terminal fragment of Yif1 is dispensable for dendrite pruning because the N-terminal deleted Yif1 transgenes completely rescued the pruning defects in Yif110-46 mutant neurons. In addition, the ddaC neurons underwent dendrite pruning normally in mutant ddaC neurons derived from a null Rab6 mutant or RNAi expression. Future work would focus on the identification of secreted molecules that trigger dendrite pruning (Wang, 2018).

This study shows that ER-Golgi transport facilitates Nrg endocytosis prior to dendrite pruning at prepupal stage. It is also possible that ER-Golgi transport directly regulates the secretion of Nrg towards the plasma membrane at larval stages. Because of a lack of an anti-Nrg antibody against its extracellular domain, this possibility could not be examined. To bypass its early role in protein secretion, Rab1DN or Sar1DN expression was induced to temporally inhibit the EG-to-Golgi transport at late larval stage, when Nrg exocytosis was completed under normal ER-to-Golgi transport. Multiple lines of evidence were provided indicating that the ER-Golgi transport facilitates Nrg endocytosis and downregulation before the onset of dendrite pruning. First, the blockade of ER-Golgi transport caused by loss of Yif1/Yip1 function or Rab1DN/Sar1DN induction leads to a significant increase of Nrg proteins in the somas, dendrites and axons, similar to that in Rab5 mutant neurons. Moreover, when the ER-Golgi transport was inhibited upon Rab1DN/Sar1DN induction, Nrg was no longer redistributed to endosomes, indicating defective Nrg endocytosis. Finally, the pruning defects caused by loss of Yif1 or Yip1 function were significantly suppressed by Nrg knockdown. It was hypothesized that, in response to activation of ecdysone signalling, some unknown cell-surface or secreted molecules might be secreted via the canonical secretory pathway to facilitate Nrg endocytosis and thereby promote dendrite pruning. Over 1000 cell-surface or secreted molecules exist in Drosophila. Future studies will be necessary to identify such secreted molecules as potential 'eat me' signals for initiating Nrg endocytosis and dendrite pruning (Wang, 2018).

L1-CAM Nrg is the only known transmembrane protein that is endocytosed during dendrite pruning in ddaC neurons. It is conceivable that ER-to-Golgi transport likely generally affects endocytosis of various transmembrane proteins. A previous study has elegantly showed that an increase of general endocytosis occurs prior to dendrite pruning; loss of Rab5 function results in blockade of general endocytosis and thereby a dendrite pruning defect. Loss of Rab5 function leads to general endocytosis defects, including the formation of enlarged Avl-positive endosomes in ddaC neurons. These Rab5 mutant phenotypes were suppressed by the blockade of ER-to-Golgi transport, suggesting that aberrant ER-to-Golgi transport generally affects endocytosis, in addition to Nrg endocytosis. Thus, ER-to-Golgi transport may also generally facilitate Rab5-dependent endocytosis during dendrite pruning of ddaC sensory neurons (Wang, 2018).

Golgi-resident Galphao promotes protrusive membrane dynamics

To form protrusions like neurites, cells must coordinate their induction and growth. The first requires cytoskeletal rearrangements at the plasma membrane (PM), the second requires directed material delivery from cell's insides. This study found that the Galphao-subunit of heterotrimeric G proteins localizes dually to PM and Golgi across phyla and cell types. The PM pool of Galphao induces, and the Golgi pool feeds, the growing protrusions by stimulated trafficking. Golgi-residing KDELR binds and activates monomeric Galphao, atypically for G protein-coupled receptors that normally act on heterotrimeric G proteins. Through multidimensional screenings identifying > 250 Galphao interactors, this study pinpoints several basic cellular activities, including vesicular trafficking, as being regulated by Galphao. It was further found small Golgi-residing GTPases Rab1 and Rab3 act as direct effectors of Galphao. This KDELR --> Galphao --> Rab1/3 signaling axis is conserved from insects to mammals and controls material delivery from Golgi to PM in various cells and tissues (Solis, 2017).

Intracellular signaling pathways currently emerge more as dynamic networks of protein interactions rather than linear cascades of activation/inactivation reactions. In this regard, thorough elucidation of the interaction targets of heterotrimeric G proteins (the immediate transducers of GPCRs) is of crucial importance to advance the understanding of this type of signal transduction. It is especially true for Gαo. Being the most abundant G protein in the nervous system and controlling multiple evolutionary conserved developmental, physiologic, and pathologic programs, it has been remarkably shy in revealing its signaling partners. This study discloses results of multiple overlapping screens, identifying > 250 interaction partners of Gαo. Each of the screens performed has its inherent advantages and limitations, and by complementation, it is thought that a near complete coverage was obtained of the Gαo interactome -- an endeavor rarely performed for a signaling protein. Cherry-picking of individual proteins from this network resulted in detailed descriptions of mechanisms of Gαo-controlled regulation of Wnt/Fz signaling, synapse formation, PCP, asymmetric cell divisions, endocytic regulation, etc., validating the interactome findings (Solis, 2017).

As opposed to characterizations of selected individual Gαo partners, this study aimed at identifying functional modules within the interactome. For this, bioinformatics analysis clustering was performed the individual components by their functions. This resulted in appearance of several major cellular activities, which now emerge to be regulated by Gαo-dependent GPCR signaling. One of them, vesicular trafficking, was selected for detailed investigation. Many important components of this cellular function, both endocytic and exocytic, are found among Gαo targets. A study previously characterized interaction of Gαo and the endocytic master regulator Rab5, important for GPCR internalization and signaling. This study now focuses more on the exocytic function of Gαo. In various cell types (neuronal, epithelial, mesenchymal) of different animal groups (insect and mammalian) this study now finds a dual localization of Gαo to Golgi and PM, and the coordinated action of the two pools is found in exocytosis and formation of various types of cellular protrusions. This study further uncovered the evolutionary conserved KDELR --> Gαo --> Rab1/Rab3 pathway at Golgi, required for stimulated material delivery to PM and the growing protrusions (Solis, 2017).

KDELR is a Golgi-residing GPCR-like receptor, activated by the cargo delivery from ER and regulating both anterograde and retrograde trafficking from Golgi. This study shows that from Drosophila to mammals, KDELR binds Gαo and activates it, potentiating Gαo-induced cellular responses. Intriguingly, this study shows that it is the βγ-free form of Gαo that is the binding and activation partner of KDELR (in a sharp contrast to the action of typical PM-localized GPCRs, which act on heterotrimeric Gαβγ complexes. It was further found that KDELR and Gαo form a multi-subunit complex, additionally containing Rab1/Rab3 GTPses and αGDI. Activation of KDELR results in the nucleotide exchange on Gαo and its dissociation from KDELR. Although recombinant Rabs interact stronger with the GTP-loaded Gαo in vitro in absence of αGDI, in cells it was found that activation of Gαo leads to dissociation of the Rab1/Rab3-αGDI complexes, ultimately resulting in activation of the small GTPases and stimulated anterograde material delivery, necessary for the growth and stabilization of cellular protrusions. Activation of KDELR is known to induce formation of multicomponent aggregates recruiting a number of additional proteins (Majoul, 2001); recruitment of Rab-GEFs to these complexes to mediate ultimate activation of Rab1/Rab3 is also conceivable but will require further investigation. Importantly, the Golgi pool of Gαo plays key roles in these processes, as the anterograde transport as well as KDELR-mediated Rab1 activation are inhibited upon depletion of Gαo (Solis, 2017).

Based on the data presented in this study, a model emerges whereas specific Gαo pools at PM and Golgi play different but cooperative roles during neuritogenesis and protrusion formation in general. At PM, Gαo initiates neurite formation regulating actin and microtubule cytoskeletons in response to activation by specific GPCRs. At Golgi, the atypical GPCR KDELR induces activation of βγ-free Gαo, which subsequently activates Rab1 and Rab3, and the combined action of these proteins potentiates the PM-directed trafficking required for elongation and stability of membrane protrusions. Being conserved from Drosophila to mammals, this molecular mechanism is of basic importance for the understanding of G protein functions in development, physiology, and disease (Solis, 2017).

The two TRAPP complexes of metazoans have distinct roles and act on different Rab GTPases

Originally identified in yeast, transport protein particle (TRAPP) complexes are Rab GTPase exchange factors that share a core set of subunits. TRAPPs were initially found to act on Ypt1, the yeast orthologue of Rab1, but recent studies have found that yeast TRAPPII can also activate the Rab11 orthologues Ypt31/32. Mammals have two TRAPP complexes, but their role is less clear, and they contain subunits that are not found in the yeast complexes but are essential for cell growth. To investigate TRAPP function in metazoans, this study shows that Drosophila melanogaster have two TRAPP complexes similar to those in mammals and that both activate Rab1, whereas one, TRAPPII, also activates Rab11. TRAPPII is not essential but becomes so in the absence of the gene parcas that encodes the Drosophila orthologue of the SH3BP5 family of Rab11 guanine nucleotide exchange factors (GEFs). Thus, in metazoans, Rab1 activation requires TRAPP subunits not found in yeast, and Rab11 activation is shared by TRAPPII and an unrelated GEF that is metazoan specific (Riedel, 2017).

Rab GTPases control many aspects of subcellular organization. They are typically active on only one particular organelle or vesicle class and so direct the subcellular localization of a wide range of proteins, including membrane traffic machinery, molecular motors, and regulators of phosphoinositide levels or the activity of other GTPases. This role in spatial organization of the cell requires specific guanine nucleotide exchange factors (GEFs) to activate each Rab in only the correct location. GEFs for several Rabs have been identified, and among the best studied are the transport protein particle (TRAPP) complexes. The first TRAPP subunit was identified in yeast in a screen for mutations that interact with a mutation in a SNARE protein, and the corresponding protein was found to be part of a large protein complex that was termed TRAPP. Subsequent work reported the existence of three different TRAPP complexes in yeast. All three share a heptameric core of six proteins (Bet3 being present twice), with TRAPPI having no further subunits, TRAPPII having four additional subunits called Tca17, Trs65, Trs120, and Trs130, and TRAPPIII having one additional subunit, Trs85. The shared TRAPP subunits are essential for membrane traffic through the Golgi apparatus, and consistent with this, TRAPPI was found to act as a GEF for Ypt1 (yeast Rab1), a GTPase essential for ER to Golgi and intra-Golgi traffic. TRAPPIII was initially reported to have a more specific role in activating Ypt1 during autophagy, but recent work suggests that TRAPPI may not exist in vivo and that TRAPPIII is responsible for the majority of Rab1 exchange activity in both secretion and autophagy. In contrast, TRAPPII was proposed to act later in the Golgi as a GEF for the closely related GTPases Ypt31 and Ypt32, yeast orthologues of Rab11. This conclusion was initially questioned, but recent biochemical studies have shown both Rab1 and Rab11 GEF activity for TRAPPII from filamentous fungi and budding yeasts (Riedel, 2017 and references therein).

The shared core TRAPP subunits that are sufficient to act on Ypt1/Rab1 are very highly conserved in evolution and appear to be a universal feature of eukaryotic cells. Mammals have orthologues of all of the yeast TRAPP subunits, including those specific to TRAPPII and TRAPPIII. In addition, coprecipitation experiments have identified two further TRAPP subunits that are not present in yeast. Examination of the proteins associated with each mammalian TRAPP subunit revealed that they form two complexes related to yeast TRAPPII and TRAPPIII, with there being no evidence that mammals have a complex equivalent to TRAPPI, i.e., just the core subunits. Mammalian TRAPPII contains seven core subunits and orthologues of Trs120 (TRAPPC9) and Trs130 (TRAPPC10). Mammalian TRAPPIII contains the same seven core subunits and an orthologue of Trs85 (TRAPPC8) plus three further subunits: TRAPPC13 (an orthologue of yeast Trs65) and the two subunits not found in yeast (TRAPPC11 and TRAPPC12) (Riedel, 2017).

The precise roles of TRAPPII and TRAPPIII in mammals are not fully resolved. When assembled in vitro, the core subunits of the mammalian TRAPP complexes have exchange activity on Rab1, and mammalian TRAPPII has been reported to have the same activity when immunoisolated from cells but to have no activity on Rab11. Moreover, there are also some striking differences to the yeast system. The most obvious is the existence of the two additional subunits in TRAPPIII, TRAPPC11 and TRAPPC12, and these seem unlikely to have minor roles as at least TRAPPC11 is essential for secretion and cell viability. In contrast, the TRAPPII subunits Trs120 and Trs130 are both essential for growth in yeast, and yet in mammals they do not appear to be required for cell viability even though Rab11 is an essential protein. Indeed, loss-of-function mutations in human TRAPPC9 are not lethal but cause mental retardation (Riedel, 2017).

The TRAPP complex subunits found in humans are well conserved across metazoans, and so this study used the tractable genetic system of Drosophila melanogaster to investigate TRAPP in metazoans. Drosophila was found to contain two TRAPP complexes that have the same composition as the human complexes, and this study combined genetics and expression of recombinant TRAPP complexes to investigate their function in vivo and their activity in vitro (Riedel, 2017).

Although recent work has shown that humans and other metazoans have two TRAPP complexes, these are not identical to their yeast counterparts. This study has provided functional evidence from Drosophila to show what is shared between metazoans and yeast and also to resolve some of the apparent paradoxes that have emerged from comparing the yeast and mammalian systems. As in yeast, both of the Drosophila TRAPP complexes have GEF activity on Rab1, whereas TRAPPII also acts as a GEF on Rab11. In the only previous study in which mammalian TRAPP complexes were tested on Rab11, TRAPPCII was immunoisolated from cells using antiserum to TRAPPC9, and activity was found on Rab1 but not Rab11. However, the subunit composition of the isolated complex was not determined, and it is also possible that the antibody or attached beads inhibited access to the Rab11 substrate. A recent study has suggested that mammalian TRAPPII can act on both Rab1 and Rab18 (Rab11 was not tested), although for reasons that are not clear, this study was unable to detect this Rab18 activity within the Drosophila complex (Riedel, 2017).

Beyond these shared properties, there are some clear differences between the yeast and metazoan TRAPP complexes. First, TRAPPIII contains two additional subunits, TRAPPC11 and TRAPPC12, that are absent from yeast. These two subunits are unlikely to have a metazoan-specific role as they are very widely conserved throughout eukaryotic phyla, including even filamentous fungi, and instead appear to have been lost in the budding yeast lineage. TRAPPC11 is distantly related to TRAPPC10, and so it seems likely that in early eukaryotic evolution, there was an ur-TRAPP complex that had two additional subunits, and then both duplicated to make on the one hand TRAPPC8 and TRAPPC9 and on the other TRAPPC10 and TRAPPC11, and hence two TRAPP complexes. TRAPPC11 is essential for the growth of both Drosophila and mammalian cultured cells, and therefore yeast must have found a means to bypass this requirement. Given that the core of shared subunits is sufficient for Rab1 GEF activity, the role of TRAPPC11 seems most likely to be to direct the TRAPPIII Rab1 GEF activity to the correct location. It is possible that yeast have evolved an alternative mechanism to direct TRAPPIII to Golgi membranes via the core subunits. However, it is also possible that TRAPPC11 allows the complex to have an additional activity that budding yeast no longer requires. The finding that in some tissues, TRAPPIII is found on the trans-Golgi as well as the cis-Golgi provides some support for this possibility as Rab1 is widely believed to function only on the cis-Golgi, and consistent with this, this study could only detect YFP-Rab1 on the cis-Golgi in these tissues. In contrast, Drosophila Rab11 is known to be present on the TGN as well as on recycling endosomes (Riedel, 2017).

This work has focused on TRAPPC11 as the TRAPPIII subunit that is essential but absent from yeast. The TRAPPIII subunit that is shared with yeast, TRAPPC8, has been reported to be essential in Drosophila and in mammalian cultured cells, although its yeast orthologue Trs85 is not essential, perhaps for the same reasons suggested in the previous pargaraph as to how yeast survive without TRAPPC11. Metazoans have two further TRAPPIII subunits: TRAPPC13, a distant orthologue of the yeast protein Trs65 that has been suggested to contribute to TRAPP complex structure, and TRAPPC12, a protein that is absent from budding yeasts but present in a diverse range of eukaryotic phyla, suggesting it had been lost during budding yeast evolution. The role of TRAPPC12 is unclear, and it is unrelated to other TRAPP subunits and has even been suggested to have additional roles outside of the TRAPP complex. TRAPPC12 has also been reported to bind to the Sec13/31 component of the COPII coat. However, although this study found this interaction when tagged TRAPPC12 was overexpressed, Sec13/31 was not recovered when TRAPPIII was precipitated with other subunits. This suggests that if this interaction is physiological, then it may represent a distinct function of TRAPPC12 or possibly an intermediate in the assembly of TRAPPIII (Riedel, 2017).

A second striking difference between the yeast and metazoan TRAPP complexes is that although TRAPPII can act on Rab11 in both classes of organism, in metazoans, it shares the role of activating Rab11 with the unrelated SH3BP5 protein family. There is clearly considerable redundancy between the two, as mutations in the SH3BP5 orthologues in Drosophila and C. elegans are viable as are mutations in TRAPPII in Drosophila and mammalian cultured cells. Loss of one or the other Rab11 GEF does have consequences, with loss of TRAPPII from flies causing male infertility and loss of Parcas causing defects in several developmental processes, some of which have been linked to signaling by nonreceptor tyrosine kinases. In C. elegans, loss of the two SH3BP5 orthologues causes defects in embryonic cytokinesis. Thus, it seems likely that each of the two types of Rab11 GEF can provide much of the necessary activation of Rab11, but each also has unique specialized roles in particular cell types. Revealing this overlap in function between the two proteins should greatly assist dissecting the function of each class of GEF. It also seems likely that TRAPPII can contribute to the activation of Rab1 in vivo as both the yeast and Drosophila complexes have activity on Rab1, at least in vitro. This could provide a possible explanation for the observation that at least in S2 cells, the major pool of TRAPPII is found on the cis-Golgi. Indeed, a study of mammalian TRAPPC10 found an epitope-tagged version of the protein to be present on the early Golgi. However, it is clear that the ability of TRAPPII to activate both Rab1 and Rab11 raises a conundrum because there is a growing consensus that Rab GEFs define the location of active Rabs within the cell. However, in this case, the two Rabs are widely believed to act on different compartments and recruit very different effector. It may be that in this case, there are additional mechanisms to restrict the recruitment of the two Rabs to a particular location before activation by TRAPP, such as a GDI displacement factor. Thus, it seems likely that further investigation of the action of the TRAPP complexes will reveal new fundamental principles of how membrane traffic is organized in cells, and this study will hopefully facilitate the pursuit of such studies in metazoan systems (Riedel, 2017).

COG7 deficiency in Drosophila generates multifaceted developmental, behavioral and protein glycosylation phenotypes

Congenital disorders of glycosylation (CDG) comprise a family of human multisystemic diseases caused by recessive mutations in genes required for protein N-glycosylation. More than 100 distinct forms of CDGs have been identified and most of them cause severe neurological impairment. The Conserved Oligomeric Golgi (COG) complex mediates tethering of vesicles carrying glycosylation enzymes across the Golgi cisternae. Mutations affecting human COG1, COG2 and COG4-COG8 cause monogenic forms of inherited, autosomal recessive CDGs. This study generated a Drosophila COG7-CDG model that closely parallels the pathological characteristics of COG7-CDG patients, including pronounced neuromotor defects associated with altered N-glycome profiles. Consistent with these alterations, larval neuromuscular junctions of Cog7 mutants exhibit a significant reduction in bouton numbers. The COG complex was shown to cooperate with Rab1 and Golgi phosphoprotein 3 to regulate Golgi trafficking; overexpression of Rab1 can rescue the cytokinesis and locomotor defects associated with loss of Cog7. These results suggest that the Drosophila COG7-CDG model can be used to test novel potential therapeutic strategies by modulating trafficking pathways (Frappaolo, 2017).

Drosophila Tempura, a novel protein prenyltransferase alpha sbunit, regulates Notch signaling via Rab1 and Rab11

Vesicular trafficking plays a key role in tuning the activity of Notch signaling. This study describes a novel and conserved Rab geranylgeranyltransferase (RabGGT)-alpha-like subunit that is required for Notch signaling-mediated lateral inhibition and cell fate determination of external sensory organs. This protein is encoded by tempura, and its loss affects the secretion of Scabrous and Delta, two proteins required for proper Notch signaling. Tempura forms a heretofore uncharacterized RabGGT complex that geranylgeranylates Rab1 and Rab11. This geranylgeranylation is required for their proper subcellular localization. A partial dysfunction of Rab1 affects Scabrous and Delta in the secretory pathway. In addition, a partial loss Rab11 affects trafficking of Delta. In summary, Tempura functions as a new geranylgeranyltransferase that regulates the subcellular localization of Rab1 and Rab11, which in turn regulate trafficking of Scabrous and Delta, thereby affecting Notch signaling (Charng, 2014).

Growing dendrites and axons differ in their reliance on the secretory pathway

Little is known about how the distinct architectures of dendrites and axons are established. From a genetic screen, this study isolated dendritic arbor reduction (dar) mutants with reduced dendritic arbors but normal axons of Drosophila neurons. dar2, dar3, and dar6 genes were identified as the homologs of Sec23, Sar1, and Rab1 of the secretory pathway. In both Drosophila and rodent neurons, defects in Sar1 expression preferentially affected dendritic growth, revealing evolutionarily conserved difference between dendritic and axonal development in the sensitivity to limiting membrane supply from the secretory pathway. Whereas limiting ER-to-Golgi transport resulted in decreased membrane supply from soma to dendrites, membrane supply to axons remained sustained. It was also shown that dendritic growth is contributed by Golgi outposts, which are found predominantly in dendrites. The distinct dependence between dendritic and axonal growth on the secretory pathway helps to establish different morphology of dendrites and axons (Ye, 2007).

In situ inhibition of vesicle transport and protein processing in the dominant negative Rab1 mutant of Drosophila

Rab proteins play an essential role in vesicle transport. In particular, RAB1 is thought to participate in the transport of most membrane and secretory proteins. To investigate the role of RAB1 in developing or functioning cells in situ, transgenic, dominant-negative Rab1 mutants of Drosophila were constructed, the protein transport and cellular and subcellular structures of mutant photoreceptor cells were examined. In the transgenic fly, the expression of mutant RAB1 was induced by Gal4 protein, whose expression was triggered by heat treatment (37 degrees C) of the fly. Within several hours after the heat induction, the lumens of the rough endoplasmic reticulum (rER) became swollen, and Golgi bodies were disassembled into vesicle clusters. Corresponding to these changes in cell structure, rhodopsin transport was blocked between the rER and the Golgi body, as indicated by the accumulation of immature rhodopsin carrying a large high-mannose-type oligosaccharide chain. Long-term expression of mutant RAB1 caused the degradation of photoreceptive microvilli and the accumulation of numerous swollen rERs, whereas no distinct changes were found in the axonal regions. These results indicate that, in Drosophila photoreceptor cells, RAB1 contributes to the maintenance of local cell structure by mediating vesicle transport between the rER and Golgi body (Satoh, 1997).


Search PubMed for articles about Drosophila Rab1

Al Awabdh, S., Miserey-Lenkei, S., Bouceba, T., Masson, J., Kano, F., Marinach-Patrice, C., Hamon, M., Emerit, M. B. and Darmon, M. (2012). A new vesicular scaffolding complex mediates the G-protein-coupled 5-HT1A receptor targeting to neuronal dendrites. J. Neurosci. 32: 14227-14241

Charng, W. L., Yamamoto, S., Jaiswal, M., Bayat, V., Xiong, B., Zhang, K., Sandoval, H., David, G., Gibbs, S., Lu, H. C., Chen, K., Giagtzoglou, N. and Bellen, H. J. (2014). Drosophila Tempura, a novel protein prenyltransferase alpha sbunit, regulates Notch signaling via Rab1 and Rab11. PLoS Biol 12: e1001777. PubMed ID: 24492843

Dippold, H. C., Ng, M. M., Farber-Katz, S. E., Lee, S. K., Kerr, M. L., Peterman, M. C., Sim, R., Wiharto, P. A., Galbraith, K. A., Madhavarapu, S., Fuchs, G. J., Meerloo, T., Farquhar, M. G., Zhou, H. and Field, S. J. (2009). GOLPH3 bridges phosphatidylinositol-4- phosphate and actomyosin to stretch and shape the Golgi to promote budding. Cell 139(2): 337-351. PubMed ID: 19837035

Frappaolo, A., Sechi, S., Kumagai, T., Robinson, S., Fraschini, R., Karimpour-Ghahnavieh, A., Belloni, G., Piergentili, R., Tiemeyer, K.H., Tiemeyer, M., Giansanti, M.G. (2017). COG7 deficiency in Drosophila generates multifaceted developmental, behavioral and protein glycosylation phenotypes. J. Cell Sci. 130(21): 3637--3649. PubMed ID: 28883096

Majoul, I., Straub, M., Hell, S. W., Duden, R. and Soling, H. D. (2001). KDEL-cargo regulates interactions between proteins involved in COPI vesicle traffic: measurements in living cells using FRET. Dev Cell 1(1): 139-153. PubMed ID: 11703931

Riedel, F., Galindo, A., Muschalik, N. and Munro, S. (2017). The two TRAPP complexes of metazoans have distinct roles and act on different Rab GTPases. J Cell Biol 217(2):601-617. PubMed ID: 29273580

Robinett, C. C., Giansanti, M. G., Gatti, M. and Fuller, M. T. (2009). TRAPPII is required for cleavage furrow ingression and localization of Rab11 in dividing male meiotic cells of Drosophila. J Cell Sci 122(Pt 24): 4526-4534. PubMed ID: 19934220

Satoh, A. K., Tokunaga, F., Kawamura, S. and Ozaki, K. (1997). In situ inhibition of vesicle transport and protein processing in the dominant negative Rab1 mutant of Drosophila. J. Cell Sci. 110: 2943-2953. 9359879

Sechi, S., Colotti, G., Belloni, G., Mattei, V., Frappaolo, A., Raffa, G. D., Fuller, M. T. and Giansanti, M. G. (2014). GOLPH3 is essential for contractile ring formation and Rab11 localization to the cleavage site during cytokinesis in Drosophila melanogaster. PLoS Genet 10(5): e1004305. PubMed ID: 24786584

Sechi, S., Frappaolo, A., Belloni, G. and Giansanti, M. G. (2015a). The roles of the oncoprotein GOLPH3 in contractile ring assembly and membrane trafficking during cytokinesis. Biochem Soc Trans 43(1): 117-121. PubMed ID: 25619256

Sechi, S., Frappaolo, A., Belloni, G., Colotti, G. and Giansanti, M. G. (2015b). The multiple cellular functions of the oncoprotein Golgi phosphoprotein 3. Oncotarget 6(6): 3493-3506. PubMed ID: 25691054

Sechi, S., Frappaolo, A., Fraschini, R., Capalbo, L., Gottardo, M., Belloni, G., Glover, D. M., Wainman, A. and Giansanti, M. G. (2017). Rab1 interacts with GOLPH3 and controls Golgi structure and contractile ring constriction during cytokinesis in Drosophila melanogaster. Open Biol 7(1). PubMed ID: 28100664

Solis, G. P., Bilousov, O., Koval, A., Luchtenborg, A. M., Lin, C. and Katanaev, V. L. (2017). Golgi-resident Galphao promotes protrusive membrane dynamics. Cell 170(5):939-955. PubMed ID: 28803726

Wang, C., Yoo, Y., Fan, H., Kim, E., Guan, K. L. and Guan, J. L. (2010). Regulation of Integrin beta 1 recycling to lipid rafts by Rab1a to promote cell migration. J Biol Chem 285(38): 29398-29405. PubMed ID: 20639577

Wang, Q., Wang, Y., Yu, F. (2018). Yif1 associates with Yip1 on Golgi and regulates dendrite pruning in sensory neurons during Drosophila metamorphosis. Development 145(12): dev164475. PubMed ID: 29769219

Wang, Y., Zhang, H., Shi, M., Liou, Y.-C., Lu, L. and Yu, F. (2017). Sec71 functions as a GEF for the small GTPase Arf1 to govern dendrite pruning of Drosophila sensory neurons. Development 144, 1851-1862

Ye, B., Zhang, Y., Song, W., Younger, S. H., Jan, L. Y. and Jan, Y. N. (2007). Growing dendrites and axons differ in their reliance on the secretory pathway. Cell 130(4): 717-729. PubMed ID: 17719548

Biological Overview

date revised: 20 November 2019

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