RAB27A

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Mitsunori Fukuda - One of the best experts on this subject based on the ideXlab platform.

  • The GTPase-deficient RAB27A(Q78L) mutant inhibits melanosome transport in melanocytes through trapping of RAB27A effector protein Slac2-a/melanophilin in their cytosol: development of a novel melanosome-targetinG tag.
    The Journal of biological chemistry, 2014
    Co-Authors: Morié Ishida, Saki P. Arai, Norihiko Ohbayashi, Mitsunori Fukuda
    Abstract:

    The small GTPase RAB27A is a crucial regulator of actin-based melanosome transport in melanocytes, and functionally defective RAB27A causes human Griscelli syndrome type 2, which is characterized by silvery hair. A GTPase-deficient, constitutively active RAB27A(Q78L) mutant has been shown to act as an inhibitor of melanosome transport and to induce perinuclear aggregation of melanosomes, but the molecular mechanism by which RAB27A(Q78L) inhibits melanosome transport remained to be determined. In this study, we attempted to identify the primary cause of the perinuclear melanosome aggregation induced by RAB27A(Q78L). The results showed that RAB27A(Q78L) is unable to localize on mature melanosomes and that its inhibitory activity on melanosome transport is completely dependent on its binding to the RAB27A effector Slac2-a/melanophilin. When we forcibly expressed RAB27A(Q78L) on mature melanosomes by using a novel melanosome-targeting tag that we developed in this study and named the MST tag, the MST-RAB27A(Q78L) fusion protein behaved in the same manner as wild-type RAB27A. It localized on mature melanosomes without inducing melanosome aggregation and restored normal peripheral melanosome distribution in RAB27A-deficient cells. These findings indicate that the GTPase activity of RAB27A is required for its melanosome localization but is not required for melanosome transport.

  • A complete Rab screening reveals novel insights in Weibel-Palade body exocytosis.
    Journal of cell science, 2012
    Co-Authors: Sofia Zografou, Dimitris Basagiannis, Alexandra Papafotika, Ryutaro Shirakawa, Hisanori Horiuchi, Daniel Auerbach, Mitsunori Fukuda, Savvas Christoforidis
    Abstract:

    Weibel-Palade bodies (WPBs) are endothelial-cell-specific organelles that, upon fusion with the plasma membrane, release cargo molecules that are essential in blood vessel abnormalities, such as thrombosis and inflammation, as well as in angiogenesis. Despite the importance of WPBs, the basic mechanisms that mediate their secretion are only poorly understood. Rab GTPases play fundamental role in the trafficking of intracellular organelles. Yet, the only known WPB-associated Rabs are RAB27A and Rab3d. To determine the full spectrum of WPB-associated Rabs we performed a complete Rab screening by analysing the localisation of all Rabs in WPBs and their involvement in the secretory process in endothelial cells. Apart from Rab3 and Rab27, we identified three additional Rabs, Rab15 (a previously reported endocytic Rab), Rab33 and Rab37, on the WPB limiting membrane. A knockdown approach using siRNAs showed that among these five WPB Rabs only Rab3, Rab27 and Rab15 are required for exocytosis. Intriguingly, we found that Rab15 cooperates with RAB27A in WPB secretion. Furthermore, a specific effector of Rab27, Munc13-4, appears to be also an effector of Rab15 and is required for WPB exocytosis. These data indicate that WPB secretion requires the coordinated function of a specific group of Rabs and that, among them, RAB27A and Rab15, as well as their effector Munc13-4, cooperate to drive exocytosis.

  • epi64 protein functions as a physiological gtpase activating protein for rab27 protein and regulates amylase release in rat parotid acinar cells
    Journal of Biological Chemistry, 2011
    Co-Authors: Akane Imai, Koutaro Ishibashi, Sumio Yoshie, Tomoko Nashida, Hiromi Shimomura, Maiko Hagatsujimura, Mitsunori Fukuda
    Abstract:

    Rab27, a small GTPase, is generally recognized as an important regulator of secretion that interacts with Rab27-specific effectors to regulate events in a wide variety of cells, including endocrine and exocrine cells. However, the mechanisms governing the spatio-temporal regulation of GTPase activity of Rab27 are not firmly established, and no GTPase-activating protein (GAP) specific for Rab27 has been identified in secretory cells. We previously showed that expression of EPI64, a Tre-2/Bub2/Cdc16 (TBC)-domain-containing protein, in melanocytes inactivates endogenous RAB27A on melanosomes (Itoh, T., and Fukuda, M. (2006) J. Biol. Chem. 281, 31823-31831), but the EPI64 role in secretory cells has never been investigated. In this study, we investigated the effect of EPI64 on Rab27 in isoproterenol (IPR)-stimulated amylase release from rat parotid acinar cells. Subcellular fractionation and immunohistochemical analyses indicated that EPI64 was enriched on the apical plasma membrane of parotid acinar cells. We found that an antibody against the TBC/Rab-GAP domain of EPI64 inhibited the reduction in levels of the endogenous GTP-Rab27 in streptolysin-O-permeabilized parotid acinar cells and suppressed amylase release in a dose-dependent manner. We also found that the levels of EPI64 mRNA and EPI64 protein increased after IPR stimulation, and that treatment with actinomycin D or antisense-EPI64 oligonucleotides suppressed the increase of EPI64 mRNA/EPI64 protein and the amount of amylase released. Our findings indicated that EPI64 acted as a physiological Rab27-GAP that enhanced GTPase activity of Rab27 in response to IPR stimulation, and that this activity is required for IPR-induced amylase release.

  • Functional characterization of two RAB27A missense mutations found in Griscelli syndrome type 2.
    Pigment cell & melanoma research, 2010
    Co-Authors: Norihiko Ohbayashi, Mitsunori Fukuda, Setareh Mamishi, Koutaro Ishibashi, Yuto Maruta, Babak Pourakbari, Banafshe Tamizifar, Masoud Mohammadpour, Nima Parvaneh
    Abstract:

    Human Griscelli syndrome type 2 (GS-2) is characterized by partial albinism and a severe immunologic disorder as a result of RAB27A mutations. In melanocytes, RAB27A forms a tripartite complex with a specific effector Slac2-a/melanophilin and myosin Va, and the complex regulates melanosome transport. Here, we report a novel homozygous missense mutation of RAB27A, i.e. K22R, in a Persian GS-2 patient and the results of analysis of the impact of the K22R mutation and the previously reported I44T mutation on protein function. Both mutations completely abolish Slac2-a/melanophilin binding activity but they affect the biochemical properties of RAB27A differently. The RAB27A(K22R) mutant lacks the GTP binding ability and exhibits cytosolic localization in melanocytes. By contrast, neither intrinsic GTPase activity nor melanosomal localization of RAB27A is affected by the I44T mutation, but the RAB27A(I44T) mutant is unable to recruit Slac2-a/melanophilin. Interestingly, the two mutations differently affect binding to other RAB27A effectors, Slp2-a, Slp4-a/granuphilin-a, and Munc13-4. The RAB27A(K22R) mutant normally binds Munc13-4, but not Slp2-a or Slp4-a, whereas the RAB27A(I44T) mutant shows reduced binding activity to Slp2-a and Munc13-4 but normally binds Slp4-a.

  • Structural basis for the exclusive specificity of Slac2-a/melanophilin for the Rab27 GTPases.
    Structure (London England : 1993), 2008
    Co-Authors: Mutsuko Kukimoto-niino, Mitsunori Fukuda, Ayako Sakamoto, Eiko Kanno, Kyoko Hanawa-suetsugu, Takaho Terada, Mikako Shirouzu, Shigeyuki Yokoyama
    Abstract:

    RAB27A is required for actin-based melanosome transport in mammalian skin melanocytes through its interaction with a specific effector, Slac2-a/melanophilin. Mutations that disrupt the RAB27A/Slac2-a interaction cause human Griscelli syndrome. The other Rab27 isoform, Rab27B, also binds all of the known effectors of RAB27A. In this study, we determined the crystal structure of the constitutively active form of Rab27B complexed with GTP and the effector domain of Slac2-a. The Rab27B/Slac2-a complex exhibits several intermolecular hydrogen bonds that were not observed in the previously reported Rab3A/rabphilin complex. A RAB27A mutation that disrupts one of the specific hydrogen bonds with Slac2-a resulted in the dramatic reduction of Slac2-a binding activity. Furthermore, we generated a Rab3A mutant that acquires Slac2-a binding ability by transplanting four Rab27-specific residues into Rab3A. These findings provide the structural basis for the exclusive association of Slac2-a with the Rab27 subfamily, whereas rabphilin binds several subfamilies, including Rab3 and Rab27.

Miguel C. Seabra - One of the best experts on this subject based on the ideXlab platform.

  • microrna containing t regulatory cell derived exosomes suppress pathogenic t helper 1 cells
    Immunity, 2014
    Co-Authors: Isobel S Okoye, Miguel C. Seabra, Tanya Tolmachova, Stephanie M Coomes, Victoria S Pelly, Stephanie Czieso, Venizelos Papayannopoulos, Mark S Wilson
    Abstract:

    Foxp3+ T regulatory (Treg) cells prevent inflammatory disease but the mechanistic basis of suppression is not understood completely. Gene silencing by RNA interference can act in a cell-autonomous and non-cell-autonomous manner, providing mechanisms of intercellular regulation. Here, we demonstrate that non-cell-autonomous gene silencing, mediated by miRNA-containing exosomes, is a mechanism employed by Treg cells to suppress T-cell-mediated disease. Treg cells transferred microRNAs (miRNA) to various immune cells, including T helper 1 (Th1) cells, suppressing Th1 cell proliferation and cytokine secretion. Use of Dicer-deficient or RAB27A and Rab27b double-deficient Treg cells to disrupt miRNA biogenesis or the exosomal pathway, respectively, established a requirement for miRNAs and exosomes for Treg-cell-mediated suppression. Transcriptional analysis and miRNA inhibitor studies showed that exosome-mediated transfer of Let-7d from Treg cell to Th1 cells contributed to suppression and prevention of systemic disease. These studies reveal a mechanism of Treg-cell-mediated suppression mediated by miRNA-containing exosomes.

  • An essential role for RAB27A GTPase in eosinophil exocytosis.
    Journal of leukocyte biology, 2013
    Co-Authors: John D. Kim, Miguel C. Seabra, Lian Willetts, Sergei I. Ochkur, Nutan Srivastava, Rudolf Hamburg, Anooshirvan Shayeganpour, James J. Lee, Redwan Moqbel, Paige Lacy
    Abstract:

    Eosinophil degranulation has been implicated in inflammatory processes associated with allergic asthma. RAB27A, a Rab-related GTPase, is a regulatory intracellular signaling molecule expressed in human eosinophils. We postulated that RAB27A regulates eosinophil degranulation. We investigated the role of RAB27A in eosinophil degranulation within the context of airway inflammation. RAB27A expression and localization in eosinophils were investigated by using subcellular fractionation combined with Western blot analysis, and the results were confirmed by immunofluorescence analysis of RAB27A and the granule membrane marker CD63. To determine the function of eosinophil RAB27A, we used Ashen mice, a strain of RAB27A-deficient animals. Ashen eosinophils were tested for degranulation in response to PAF and calcium ionophore by measuring released EPX activity. Airway EPX release was also determined by intratracheal injection of eosinophils into mice lacking EPX. RAB27A immunoreactivity colocalized with eosinophil crystalloid granules, as determined by subcellular fractionation and immunofluorescence analysis. PAF induced eosinophil degranulation in correlation with redistribution of RAB27A(+) structures, some of which colocalized with CD63(+) crystalloid granules at the cell membrane. Eosinophils from mice had significantly reduced EPX release compared with normal WT eosinophils, both in vitro and in vivo. In mouse models, Ashen mice demonstrated reduced EPX release in BAL fluid. These findings suggest that RAB27A has a key role in eosinophil degranulation. Furthermore, these findings have implications for RAB27A-dependent eosinophil degranulation in airway inflammation.

  • RAB27A supports exosome dependent and independent mechanisms that modify the tumor microenvironment and can promote tumor progression
    Cancer Research, 2012
    Co-Authors: Angelique Bobrie, Miguel C. Seabra, Matias Ostrowski, Sophie Krumeich, Chiara Recchi, Fabien Reyal, Luis F Moita, Clotilde Thery
    Abstract:

    During progression from single cancer cells to a tumor mass and metastases, tumor cells send signals that can subvert their tissue microenvironment. These signals involve soluble molecules and various extracellular vesicles, including a particular type termed exosomes. The specific roles of exosomes secreted in the tumor microenvironment, however, is unclear. The small GTPases RAB27A and RAB27B regulate exocytosis of multivesicular endosomes, which lead to exosome secretion, in human HeLa cells. Here, we used mouse models to show that RAB27A blockade in mammary carcinoma cells decreased secretion of exosomes characterized by endocytic markers, but also of matrix metalloproteinase 9, which is not associated with exosomes. RAB27A blockade resulted in decreased primary tumor growth and lung dissemination of a metastatic carcinoma (4T1), but not of a nonmetastatic carcinoma (TS/A). Local growth of 4T1 tumors required mobilization of a population of neutrophil immune cells induced by RAB27A-dependent secretion of exosomes together with a specific combination of cytokines and/or metalloproteinases. Our findings offer in vivo validation of the concept that exosome secretion can exert key pathophysiologic roles during tumor formation and progression, but they also highlight the idiosyncratic character of the tumor context.

  • RAB27A Targeting to Melanosomes Requires Nucleotide Exchange but Not Effector Binding
    Traffic (Copenhagen Denmark), 2011
    Co-Authors: Abul K Tarafder, Jose S Ramalho, Christina Wasmeier, Alistair N. Hume, Ana C. Figueiredo, Antonia E. G. Booth, Asumi Orihara, Miguel C. Seabra
    Abstract:

    Rab GTPases are important determinants of organelle identity and regulators of vesicular transport pathways. Consequently, each Rab occupies a highly specific subcellular localization. However, the precise mechanisms governing Rab targeting remain unclear. Guanine nucleotide exchange factors (GEFs), putative membrane-resident targeting factors and effector binding have all been implicated as critical regulators of Rab targeting. Here, we address these issues using RAB27A targeting to melanosomes as a model system. RAB27A regulates motility of lysosome-related organelles and secretory granules. Its effectors have been characterized extensively, and we have identified Rab3GEP as the non-redundant RAB27A GEF in melanocytes (Figueiredo AC et al. Rab3GEP is the non-redundant guanine nucleotide exchange factor for RAB27A in melanocytes. J Biol Chem 2008;283:23209–23216). Using RAB27A mutants that show impaired binding to representatives of all four RAB27A effector subgroups, we present evidence that effector binding is not essential for targeting of RAB27A to melanosomes. In contrast, we observed that knockdown of Rab3GEP resulted in mis-targeting of RAB27A, suggesting that Rab3GEP activity is required for correct targeting of RAB27A. However, the identification of RAB27A mutants that undergo efficient GDP/GTP exchange in the presence of Rab3GEP in vitro but are mis-targeted in a cellular context indicates that nucleotide loading is not the sole determinant of subcellular targeting of RAB27A. Our data support a model in which exchange activity, but not effector binding, represents one essential factor that contributes to membrane targeting of Rab proteins.

  • RAB27A and Rab27b regulate neutrophil azurophilic granule exocytosis and NADPH oxidase activity by independent mechanisms.
    Traffic (Copenhagen Denmark), 2009
    Co-Authors: Jennifer L. Johnson, Miguel C. Seabra, Agnieszka A. Brzezinska, Tanya Tolmachova, Daniela B. Munafo, Beverly A. Ellis, Hong Hong, Sergio D. Catz
    Abstract:

    Neutrophils rely on exocytosis to mobilize receptors and adhesion molecules and to release microbicidal factors. This process should be strictly regulated because uncontrolled release of toxic proteins would be injurious to the host. In vivo studies showed that the small GTPase RAB27A regulates azurophilic granule exocytosis. Using mouse neutrophils deficient in RAB27A (RAB27Aash/ash), Rab27b [Rab27b knockout (KO)] or both [RAB27A/b double KO (DoKo)], we investigated the role of the Rab27 isoforms in neutrophils. We found that both RAB27A and Rab27b deficiencies impaired azurophilic granule exocytosis. RAB27Aash/ash neutrophils showed upregulation of Rab27b expression which did not compensate for the secretory defects observed in RAB27A-deficient cells, suggesting that Rab27 isoforms play independent roles in neutrophil exocytosis. Total internal reflection fluorescence microscopy analysis showed that RAB27Aash/ash and Rab27b KO neutrophils have a decreased number of azurophilic granules near the plasma membrane. The effect was exacerbated in RAB27A/b DoKo neutrophils. Rab27-deficient neutrophils showed impaired activation of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase at the plasma membrane although intraphagosomal reactive oxygen species (ROS) production was not affected. Exocytosis of secretory vesicles in Rab27-deficient neutrophils was functional, suggesting that Rab27 GTPases selectively control the exocytosis of neutrophil granules.

Tetsuro Izumi - One of the best experts on this subject based on the ideXlab platform.

  • Rab2a and RAB27A cooperatively regulate the transition from granule maturation to exocytosis through the dual effector Noc2.
    Journal of cell science, 2016
    Co-Authors: Kohichi Matsunaga, Masato Taoka, Toshiaki Isobe, Tetsuro Izumi
    Abstract:

    Exocytosis of secretory granules entails budding from the trans-Golgi network, sorting and maturation of cargo proteins, and trafficking and fusion to the plasma membrane. RAB27A regulates the late steps in this process, such as granule recruitment to the fusion site, whereas Rab2a functions in the early steps, such as granule biogenesis and maturation. Here, we demonstrate that these two small GTPases simultaneously bind to Noc2 (also known as RPH3AL) in a GTP-dependent manner, although Rab2a binds only after RAB27A has bound. In pancreatic β-cells, the ternary Rab2a-Noc2-RAB27A complex specifically localizes on perinuclear immature granules, whereas the binary Noc2-RAB27A complex localizes on peripheral mature granules. In contrast to the wild type, Noc2 mutants defective in binding to Rab2a or RAB27A fail to promote glucose-stimulated insulin secretion. Although knockdown of any component of the ternary complex markedly inhibits insulin secretion, only knockdown of Rab2a or Noc2, and not that of RAB27A, impairs cargo processing from proinsulin to insulin. These results suggest that the dual effector, Noc2, regulates the transition from Rab2a-mediated granule biogenesis to RAB27A-mediated granule exocytosis.

  • Elucidation of Rab27 Recruitment by Its Effectors: Structure of RAB27A Bound to Exophilin4/Slp2-a
    Structure (London England : 1993), 2008
    Co-Authors: Leonard M. G. Chavas, Seiji Torii, Tetsuro Izumi, Masato Kawasaki, Kentaro Ihara, Ryuichi Kato, Tamami Uejima, Soichi Wakatsuki
    Abstract:

    Summary Rab GTPases coordinate vesicular trafficking within eukaryotic cells by collaborating with a set of effector proteins. RAB27A regulates numerous exocytotic pathways, and its dysfunction causes the Griscelli syndrome human immunodeficiency. Exophilin4/Slp2-a localizes on phosphatidylserine-enriched plasma membrane, and its N-terminal Rab27-binding domain (RBD27) specifically recognizes Rab27 on the surfaces of melanosomes and secretory granules prior to docking and fusion. To characterize the selective binding of Rab27 to 11 various effectors, we have determined the 1.8 A resolution structure of RAB27A in complex with Exophilin4 RBD27. The effector packs against the switch and interswitch elements of RAB27A, and specific affinity toward RAB27A is modulated by a shift in the orientation of the effector structural motif ( S /T)( G /L) xW ( F /Y) 2 . The observed structural complementation between the interacting surfaces of RAB27A and Exophilin4 sheds light on the disparities among the Rab27 effectors and outlines a general mechanism for their recruitment.

  • elucidation of rab27 recruitment by its effectors structure of RAB27A bound to exophilin4 slp2 a
    Structure, 2008
    Co-Authors: Seiji Torii, Tetsuro Izumi, Leonard M. G. Chavas, Masato Kawasaki, Kentaro Ihara, Ryuichi Kato, Tamami Uejima, Soichi Wakatsuki
    Abstract:

    Summary Rab GTPases coordinate vesicular trafficking within eukaryotic cells by collaborating with a set of effector proteins. RAB27A regulates numerous exocytotic pathways, and its dysfunction causes the Griscelli syndrome human immunodeficiency. Exophilin4/Slp2-a localizes on phosphatidylserine-enriched plasma membrane, and its N-terminal Rab27-binding domain (RBD27) specifically recognizes Rab27 on the surfaces of melanosomes and secretory granules prior to docking and fusion. To characterize the selective binding of Rab27 to 11 various effectors, we have determined the 1.8 A resolution structure of RAB27A in complex with Exophilin4 RBD27. The effector packs against the switch and interswitch elements of RAB27A, and specific affinity toward RAB27A is modulated by a shift in the orientation of the effector structural motif ( S /T)( G /L) xW ( F /Y) 2 . The observed structural complementation between the interacting surfaces of RAB27A and Exophilin4 sheds light on the disparities among the Rab27 effectors and outlines a general mechanism for their recruitment.

  • Purification, crystallization and preliminary X-ray crystallographic analysis of RAB27A GTPase in complex with exophilin4/Slp2-a effector.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2008
    Co-Authors: Leonard M. G. Chavas, Tetsuro Izumi, Masato Kawasaki, Kentaro Ihara, Ryuichi Kato, Soichi Wakatsuki
    Abstract:

    By switching between GTP-active and GDP-inactive conformations, small Ras GTPases partly regulate membrane trafficking, cell growth and cytoskeleton dynamics. Among Rab GTPases, the Rab27 subfamily, which comprises RAB27A and Rab27b, controls the proper targeting of secretory vesicles to the plasma membrane. GppNHp-bound RAB27A in complex with the Rab27-binding domain of exophilin4/Slp2-a effector has been purified and crystallized for structural studies. The crystals belong to space group P212121 and a complete data set was collected to a resolution of 1.8 A. Eventually, the structural characterization of the RAB27A–exophilin4/Slp2-a complex will clarify Rab27 recognition by its effectors prior to vesicle tethering and docking.

  • rab27b is expressed in a wide range of exocytic cells and involved in the delivery of secretory granules near the plasma membrane
    Molecular Biology of the Cell, 2007
    Co-Authors: Hiroshi Gomi, Kenichi Mori, Shigeyoshi Itohara, Tetsuro Izumi
    Abstract:

    Rab proteins regulate multiple, complex processes of membrane traffic. Among these proteins, RAB27A has been shown to function specifically in regulated exocytic pathways. However, the roles of Rab27b, another Rab27 subfamily member, have not been well characterized. We disrupted the Rab27b gene in mice. The targeting vector was designed to insert LacZ downstream of the initiation codon of the Rab27b gene so that the authentic promoter should drive this reporter gene. A comprehensive analysis of Rab27b expression using this mouse strain indicated that it is widely expressed not only in canonical secretory cells, but also in neurons and cells involved in surface protection and mechanical extension. To evaluate the function in pituitary endocrine cells where the isoform RAB27A is coexpressed, we generated RAB27A/Rab27b double knockout mice by crossing Rab27b knockout mice with RAB27A-mutated ashen mice. The polarized distribution of secretory granules close to the plasma membrane was markedly impaired in the pituitary of double knockout mice, indicating that the Rab27 subfamily is involved in the delivery of granules near the exocytic site. In conjunction with a phenotype having a pituitary devoid of the Rab27 effector granuphilin, we discuss the relationship between the residence and the releasable pool of granules.

Alistair N. Hume - One of the best experts on this subject based on the ideXlab platform.

  • distinct and opposing roles for RAB27A mlph myova and rab27b munc13 4 in mast cell secretion
    FEBS Journal, 2012
    Co-Authors: Rajesh K Singh, Christina Wasmeier, Tanya Tolmachova, Sergio D. Catz, Koichi Mizuno, Silene T Wavreshapton, Chiara Recchi, Clare E Futter, Alistair N. Hume
    Abstract:

    Mediator release from mast cells is a critical step in allergic and inflammatory disease. However, the processes regulating the latter stages of granule release are yet to be fully understood. Rab27 small GTPases regulate release of secretory lysosomes in a variety of cells, including mast cell granules. In the present study, using murine bone marrow-derived mast cells (BMMC) from Rab27-deficient mutant mice, we found that, in contrast to Rab27b, RAB27A primarily plays an inhibitory role in regulating degranulation. Immunofluorescence analysis revealed that resting RAB27A-deficient (ashen) BMMCs display abnormal cortical F-actin distribution. Actin disassembly prior to IgE cross-linking increased wild-type BMMC secretion to ashen levels, suggesting that changes in the integrity of cortical F-actin underlie the ashen phenotype. Comparison of the secretory impairment of Rab27b knockout and RAB27A/b double knockout BMMCs highlighted a secondary positive role for RAB27A in enhancing degranulation. Rab27 is known to interact with actin via its effectors melanophilin (Mlph) and myosin Va (MyoVa) in other cell types. To better understand the differing roles of Rab27 proteins, we analysed the secretory phenotype of BMMCs derived from mice lacking Rab27 effector proteins. These experiments revealed that the phenotype of BMMCs deficient in Mlph (leaden) and BMMCs deficient in MyoVa (dilute) resembles the hyper-secretion of ashen BMMCs, while Munc13-4-deficient (jinx) BMMCs phenocopy the Rab27b knockout and double RAB27A/b knockout secretory impairment. We conclude that RAB27A and Rab27b regulate distinct steps in the BMMC degranulation pathway, with RAB27A/Mlph/MyoVa regulating cortical actin stability upstream of RAB27A/b/Munc13-4-dependent granule exocytosis.

  • RAB27A Targeting to Melanosomes Requires Nucleotide Exchange but Not Effector Binding
    Traffic (Copenhagen Denmark), 2011
    Co-Authors: Abul K Tarafder, Jose S Ramalho, Christina Wasmeier, Alistair N. Hume, Ana C. Figueiredo, Antonia E. G. Booth, Asumi Orihara, Miguel C. Seabra
    Abstract:

    Rab GTPases are important determinants of organelle identity and regulators of vesicular transport pathways. Consequently, each Rab occupies a highly specific subcellular localization. However, the precise mechanisms governing Rab targeting remain unclear. Guanine nucleotide exchange factors (GEFs), putative membrane-resident targeting factors and effector binding have all been implicated as critical regulators of Rab targeting. Here, we address these issues using RAB27A targeting to melanosomes as a model system. RAB27A regulates motility of lysosome-related organelles and secretory granules. Its effectors have been characterized extensively, and we have identified Rab3GEP as the non-redundant RAB27A GEF in melanocytes (Figueiredo AC et al. Rab3GEP is the non-redundant guanine nucleotide exchange factor for RAB27A in melanocytes. J Biol Chem 2008;283:23209–23216). Using RAB27A mutants that show impaired binding to representatives of all four RAB27A effector subgroups, we present evidence that effector binding is not essential for targeting of RAB27A to melanosomes. In contrast, we observed that knockdown of Rab3GEP resulted in mis-targeting of RAB27A, suggesting that Rab3GEP activity is required for correct targeting of RAB27A. However, the identification of RAB27A mutants that undergo efficient GDP/GTP exchange in the presence of Rab3GEP in vitro but are mis-targeted in a cellular context indicates that nucleotide loading is not the sole determinant of subcellular targeting of RAB27A. Our data support a model in which exchange activity, but not effector binding, represents one essential factor that contributes to membrane targeting of Rab proteins.

  • loss of rab27 function results in abnormal lung epithelium structure in mice
    American Journal of Physiology-cell Physiology, 2011
    Co-Authors: Giulia Bolasco, Tanya Tolmachova, Dhani Traceywhite, Andrew Thorley, Teresa D Tetley, Michael C Seabra, Alistair N. Hume
    Abstract:

    Rab27 small GTPases regulate secretion and movement of lysosome-related organelles such as T cell cytolytic granules and platelet-dense granules. Previous studies indicated that RAB27A and Rab27b are expressed in the murine lung suggesting that they regulate secretory processes in the lung. Consistent with those studies, we found that RAB27A and Rab27b are expressed in cell types that contain secretory granules: alveolar epithelial type II (AEII) and Clara cells. We then used RAB27A/Rab27b double knockout (DKO) mice to examine the functional consequence of loss of Rab27 proteins in the murine lung. Light and electron microscopy revealed a number of morphological changes in lungs from DKO mice when compared with those in control animals. In aged DKO mice we observed atrophy of the bronchiolar and alveolar epithelium with reduction of cells numbers, thinning of the bronchiolar epithelium and alveolar walls, and enlargement of alveolar airspaces. In these samples we also observed increased numbers of activated foamy alveolar macrophages and granulocyte containing infiltrates together with reduction in the numbers of Clara cells and AEII cells compared with control. At the ultrastructural level we observed accumulation of cytoplasmic membranes and vesicles in Clara cells. Meanwhile, AEII cells in DKO accumulated large mature lamellar bodies and lacked immature/precursor lamellar bodies. We hypothesize that the morphological changes observed at the ultrastructural level in DKO samples result from secretory defects in AEII and Clara cells and that over time these defects lead to atrophy of the epithelium.

  • RAB27A and Rab27b control different steps of the exosome secretion pathway
    Nature Cell Biology, 2010
    Co-Authors: Matias Ostrowski, Graca Raposo, Sophie Krumeich, Isabelle Fanget, Ariel Savina, Catarina Moita, Alistair N. Hume, Nuno B. Carmo, Kristine Schauer, Rui P. Freitas
    Abstract:

    Exosomes are secreted membrane vesicles that share structural and biochemical characteristics with intraluminal vesicles of multivesicular endosomes (MVEs). Exosomes could be involved in intercellular communication and in the pathogenesis of infectious and degenerative diseases. The molecular mechanisms of exosome biogenesis and secretion are, however, poorly understood. Using an RNA interference (RNAi) screen, we identified five Rab GTPases that promote exosome secretion in HeLa cells. Among these, RAB27A and Rab27b were found to function in MVE docking at the plasma membrane. The size of MVEs was strongly increased by RAB27A silencing, whereas MVEs were redistributed towards the perinuclear region upon Rab27b silencing. Thus, the two Rab27 isoforms have different roles in the exosomal pathway. In addition, silencing two known Rab27 effectors, Slp4 (also known as SYTL4, synaptotagmin-like 4) and Slac2b (also known as EXPH5, exophilin 5), inhibited exosome secretion and phenocopied silencing of RAB27A and Rab27b, respectively. Our results therefore strengthen the link between MVEs and exosomes, and introduce ways of manipulating exosome secretion in vivo . Exosome biogenesis is poorly understood. The small GTPases RAB27A and Rab27b and their effectors, Slp4 and Slac2b, control exosome secretion at different steps by regulating the peripheral localization, retention and docking of exosomal precursors, the multivesicular endosomes.

  • a family of rab27 binding proteins melanophilin links RAB27A and myosin va function in melanosome transport
    Journal of Biological Chemistry, 2002
    Co-Authors: Molly Strom, Alistair N. Hume, Abul K Tarafder, Eleni Barkagianni, Miguel C. Seabra
    Abstract:

    The RAB27A GTPase regulates diverse processes involving lysosome-related organelles, including melanosome motility in melanocytes, and lytic granule release in cytotoxic T lymphocytes. Toward an understanding of RAB27A function, we searched for proteins that interact with RAB27A(GTP) using the yeast two-hybrid system and identified JFC1/Slp1, a protein of unknown function. JFC1/Slp1 and related proteins, including melanophilin, contain a conserved amino-terminal domain similar to the Rab3a-binding domain of Rabphilin-3. We used several methods to demonstrate that this conserved amino-terminal domain is a Rab27-binding domain. We show that this domain interacts directly, and in a GTP-dependent manner with RAB27A. Furthermore, overexpression of this domain in melanocytes results in perinuclear clustering of melanosomes, suggesting that this region is sufficient for interaction with, and perturbation of function of, RAB27A in a physiological context. Thus, we identified a novel family of Rab27-binding proteins. We also show that melanophilin associates with RAB27A and myosin Va on melanosomes in melanocytes, and present evidence that a domain within the carboxyl-terminal region of melanophilin interacts with the carboxyl-terminal tail of the melanocyte-specific splice isoform of myosin Va. Thus, melanophilin can associate simultaneously with activated RAB27A and myosin Va via distinct regions, and serve as a linker between these proteins.

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  • Elucidation of Rab27 Recruitment by Its Effectors: Structure of RAB27A Bound to Exophilin4/Slp2-a
    Structure (London England : 1993), 2008
    Co-Authors: Leonard M. G. Chavas, Seiji Torii, Tetsuro Izumi, Masato Kawasaki, Kentaro Ihara, Ryuichi Kato, Tamami Uejima, Soichi Wakatsuki
    Abstract:

    Summary Rab GTPases coordinate vesicular trafficking within eukaryotic cells by collaborating with a set of effector proteins. RAB27A regulates numerous exocytotic pathways, and its dysfunction causes the Griscelli syndrome human immunodeficiency. Exophilin4/Slp2-a localizes on phosphatidylserine-enriched plasma membrane, and its N-terminal Rab27-binding domain (RBD27) specifically recognizes Rab27 on the surfaces of melanosomes and secretory granules prior to docking and fusion. To characterize the selective binding of Rab27 to 11 various effectors, we have determined the 1.8 A resolution structure of RAB27A in complex with Exophilin4 RBD27. The effector packs against the switch and interswitch elements of RAB27A, and specific affinity toward RAB27A is modulated by a shift in the orientation of the effector structural motif ( S /T)( G /L) xW ( F /Y) 2 . The observed structural complementation between the interacting surfaces of RAB27A and Exophilin4 sheds light on the disparities among the Rab27 effectors and outlines a general mechanism for their recruitment.

  • elucidation of rab27 recruitment by its effectors structure of RAB27A bound to exophilin4 slp2 a
    Structure, 2008
    Co-Authors: Seiji Torii, Tetsuro Izumi, Leonard M. G. Chavas, Masato Kawasaki, Kentaro Ihara, Ryuichi Kato, Tamami Uejima, Soichi Wakatsuki
    Abstract:

    Summary Rab GTPases coordinate vesicular trafficking within eukaryotic cells by collaborating with a set of effector proteins. RAB27A regulates numerous exocytotic pathways, and its dysfunction causes the Griscelli syndrome human immunodeficiency. Exophilin4/Slp2-a localizes on phosphatidylserine-enriched plasma membrane, and its N-terminal Rab27-binding domain (RBD27) specifically recognizes Rab27 on the surfaces of melanosomes and secretory granules prior to docking and fusion. To characterize the selective binding of Rab27 to 11 various effectors, we have determined the 1.8 A resolution structure of RAB27A in complex with Exophilin4 RBD27. The effector packs against the switch and interswitch elements of RAB27A, and specific affinity toward RAB27A is modulated by a shift in the orientation of the effector structural motif ( S /T)( G /L) xW ( F /Y) 2 . The observed structural complementation between the interacting surfaces of RAB27A and Exophilin4 sheds light on the disparities among the Rab27 effectors and outlines a general mechanism for their recruitment.

  • Purification, crystallization and preliminary X-ray crystallographic analysis of RAB27A GTPase in complex with exophilin4/Slp2-a effector.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2008
    Co-Authors: Leonard M. G. Chavas, Tetsuro Izumi, Masato Kawasaki, Kentaro Ihara, Ryuichi Kato, Soichi Wakatsuki
    Abstract:

    By switching between GTP-active and GDP-inactive conformations, small Ras GTPases partly regulate membrane trafficking, cell growth and cytoskeleton dynamics. Among Rab GTPases, the Rab27 subfamily, which comprises RAB27A and Rab27b, controls the proper targeting of secretory vesicles to the plasma membrane. GppNHp-bound RAB27A in complex with the Rab27-binding domain of exophilin4/Slp2-a effector has been purified and crystallized for structural studies. The crystals belong to space group P212121 and a complete data set was collected to a resolution of 1.8 A. Eventually, the structural characterization of the RAB27A–exophilin4/Slp2-a complex will clarify Rab27 recognition by its effectors prior to vesicle tethering and docking.

  • Structure of the small GTPase Rab27b shows an unexpected swapped dimer
    Acta Crystallographica Section D Biological Crystallography, 2007
    Co-Authors: Leonard M. G. Chavas, Seiji Torii, Tetsuro Izumi, Hironari Kamikubo, Masato Kawasaki, Kentaro Ihara, Ryuichi Kato, Mikio Kataoka, Soichi Wakatsuki
    Abstract:

    Members of the Rab family of small GTPases regulate membrane traffic within the cell by recruiting their specific effectors in a nucleotide-dependent manner. The Rab27 subfamily consists of RAB27A and Rab27b, which share 70% sequence identity. By interacting with a large set of effector proteins such as melanophilin and granuphilin, both RAB27A and Rab27b regulate the exocytosis of secretory lysosomes. Here, the crystal structures of mouse Rab27b in complex with GDP have been determined in three distinct crystal lattices. Surprisingly, Rab27b-GDP exists in an open conformation with protruding switch and interswitch regions, which are stabilized through dimerization by means of domain-swapping in the crystals. In contrast, small-angle X-ray scattering measurements showed an extended monomer form of Rab27b in solution. The observed dimer formation of Rab27b-GDP in the crystals would restrain the highly flexible switch regions. Possible biological implications of this atypical structure of Rab27b and its plausible influence in effector interaction are discussed.