Constitutive GDP/GTP exchange and secretion-dependent GTP hydrolysis activity for Rab27 in platelets

We have previously demonstrated that Rab27 regulates dense granule secretion in platelets. Here, we analyzed the activation status of Rab27 using the thin layer chromatography method analyzing nucleotides bound to immunoprecipitated Rab27 and the pull-down method quantifying Rab27 bound to the GTP-Rab27-binding domain (synaptotagmin-like protein (Slp)-homology domain) of its specific effector, Slac2-b. We found that Rab27 was predominantly present in the GTP-bound form in unstimulated platelets due to constitutive GDP/GTP exchange activity. The GTP-bound Rab27 level drastically decreased due to enhanced GTP hydrolysis activity upon granule secretion. In permeabilized platelets, increase of Ca(2+) concentration induced dense granule secretion with concomitant decrease of GTP-Rab27, whereas in non-hydrolyzable GTP analogue GppNHp (beta-gamma-imidoguanosine 5'-triphosphate)-loaded permeabilized platelets, the GTP (GppNHp)-Rab27 level did not decrease upon the Ca(2+)-induced secretion. These data suggested that GTP hydrolysis of Rab27 was not necessary for inducing the secretion. Taken together, Rab27 is maintained in the active status in unstimulated platelets, which could function to keep dense granules in a preparative status for secretion.     

Involvement of Rab3A in vesicle priming during exocytosis: interaction with Munc13-1 and Munc18-1

Rab3A is a small G-protein of the Rab family that is involved in the late steps of exocytosis. Here, we studied the role of Rab3A and its relationship with Munc13-1 and Munc18-1 during vesicle priming. Phorbol 12-myristate 13-acetate (PMA) is known to enhance the percentage of fusion-competent vesicles and this is mediated by protein kinase C (PKC)-independent Munc13-1 activation and PKC-dependent dissociation of Munc18-1 from syntaxin 1a. Our results show that the effects of PMA varied in cells overexpressing Rab3A or mutants of Rab3A and in cells with Rab3A knockdown. When Munc13-1 was overexpressed in Rab3A knockdown cells, secretion was completely inhibited. In cells overexpressing a Rab-interacting molecule (RIM)-binding deficient Munc13-1 mutant, 128-Munc13-1, the effects of Rab3A on PMA-induced secretion was abolished. The effect of PMA, which disappeared in cells overexpressing GTP-Rab3A (Q81L), could be reversed by co-expressing Munc18-1 but not its mutant R39C, which is unable to bind to syntaxin 1a. In cells overexpressing Munc18-1, manipulation of Rab3A activity had no effect on secretion. Finally, Munc18-1 enhanced the dissociation of Rab3A, and such enhancement correlated with exocytosis. In summary, our results support the hypothesis that the Rab3A cycle is coupled with the activation of Munc13-1 via RIM, which accounts for the regulation of secretion by Rab3A. Munc18-1 acts downstream of Munc13-1/RIM/Rab3A and interacts with syntaxin 1a allowing vesicle priming. Furthermore, Munc18-1 promotes Rab3A dissociation from vesicles, which then results in fusion.     

The Rab-binding protein Noc2 is associated with insulin-containing secretory granules and is essential for pancreatic beta-cell exocytosis

The small GTPases Rab3 and Rab27 are associated with secretory granules of pancreatic beta-cells and regulate insulin exocytosis. In this study, we investigated the role of Noc2, a potential partner of these two GTPases, in insulin secretion. In the beta-cell line INS-1E wild-type Noc2, Noc265E, and Noc258A, a mutant capable of interacting with Rab27 but not Rab3, colocalized with insulin-containing vesicles. In contrast, two mutants (Noc2138S,141S and Noc2154A,155A,156A) that bind neither Rab3 nor Rab27 did not associate with secretory granules and were uniformly distributed throughout the cell cytoplasm. Overexpression of wild-type Noc2, Noc265E, or Noc258A inhibited hormone secretion elicited by insulin secretagogues. In contrast, overexpression of the mutants not targeted to secretory granules was without effect. Silencing of the Noc2 gene by RNA interference led to a strong impairment in the capacity of INS-1E cells to respond to insulin secretagogues, indicating that appropriate levels of Noc2 are essential for pancreatic beta-cell exocytosis. The defect was already detectable in the early secretory phase (0-10 min) but was particularly evident during the sustained release phase (10-45 min). Protein-protein binding studies revealed that Noc2 is a potential partner of Munc13, a component of the machinery that controls vesicle priming and insulin exocytosis. These data suggest that Noc2 is involved in the recruitment of secretory granules at the plasma membrane possibly via the interaction with Munc13.     

Rab27a regulates epithelial sodium channel (ENaC) activity through synaptotagmin-like protein (SLP-5) and Munc13-4 effector mechanism

Liddle's syndrome (excessive absorption of sodium ions) and PHA-1 (pseudohypoaldosteronism type 1) with decreased sodium absorption are caused by the mutations in the amiloride-sensitive epithelial sodium channel ENaC. Rab proteins are small GTPases involved in vesicle transport, docking, and fusion. Earlier, we reported that Rab27a inhibits ENaC-mediated currents through protein-protein interaction in HT-29 cells. We hereby report that Rab27a-dependent inhibition is associated with the GTP/GDP status as constitutively active or GTPase-deficient mutant Q78L inhibits amiloride-sensitive currents whereas GDP-locked inactive mutant T23N showed no effect. In order to further explore the molecular mechanism of this regulation, we performed competitive assays with two Rab27a-binding proteins: synaptotagmin-like protein (SLP-5) and Munc13-4 (a putative priming factor for exocytosis). Both proteins eliminate negative modulation of Rab27a on ENaC function. The SLP-5 reversal of Rab27a effect was restricted to C-terminal C2A/C2B domains assigned for putative phospholipids-binding function while the Rab27a-binding SHD motif imparted higher inhibition. The ENaC-mediated currents remain unaffected by Rab27a though SLP-5 appears to strongly bind it. The immunoprecipitation experiments suggest that in the presence of excessive Munc13-4 and SLP-5 proteins, Rab27a interaction with ENaC is diminished. Munc13-4 and SLP-5 limit the Rab27a availability to ENaC, thus minimizing its effect on channel function. These observations decisively prove that Rab27a inhibits ENaC function through a complex mechanism that involves GTP/GDP status, and protein-protein interactions involving Munc13-4 and SLP-5 effector proteins.     

Involvement of Rab27 in antigen-induced histamine release from rat basophilic leukemia 2H3 cells

The Rab family small G proteins regulate discrete steps in vesicular transport pathways. Recent studies indicate that one member of the Rab family, Rab27A, regulates the transport of lysosome-related organelles, such as melanosome distribution in melanocytes, lytic granule release in cytotoxic T cells, and dense granule release in platelets. Here, we have examined the involvement of Rab27A in the exocytic transport of another lysosome-related organelle, the basophilic secretory granule, in basophils. We have found that Rab27A locates on basophilic secretory granules containing histamine in rat basophilic leukemia (RBL) 2H3 cells. In addition, exogenous expression of dominant active Rab27A reduces antigen-induced histamine release from the cells. We have moreover identified Munc13-4 as a Rab27A target using a CytoTrap system and found that exogenous expression of Munc13-4 affects antigen-induced histamine release from RBL-2H3 cells. These results demonstrate that Rab27A plays a crucial role in antigen-induced histamine release from RBL-2H3 cells.     

The Rab27a effectors JFC1/Slp1 and Munc13-4 regulate exocytosis of neutrophil granules

Neutrophil granules contain secretory molecules that contribute to the implementation of all neutrophil functions. The molecular components that regulate the exocytosis of neutrophil granules have not been characterized. In this study, using small interfering RNA gene-targeting approaches and granulocytes from genetically modified mice, we characterized the Rab27a effectors JFC1/Slp1 and Munc13-4 as components of the exocytic machinery of granulocytes. Using total internal reflection fluorescence microscopy analysis, we show that Rab27a and JFC1 colocalize in predocked and docked vesicles in granulocytes. Next, we demonstrate that JFC1-downregulated granulocytes have impaired myeloperoxidase secretion. Using immunological interference, we confirm that JFC1 plays an important role in azurophilic granule exocytosis in human neutrophils. Interference with Rab27a but not with JFC1 impaired gelatinase B secretion in neutrophils, suggesting that a different Rab27a effector modulates this process. In similar studies, we confirmed that Munc13-4 regulates gelatinase secretion. Immunofluorescence analysis indicates that Munc13-4 localizes at secretory organelles in neutrophils. Using neutrophils from a Munc13-4-deficient mouse model (Jinx), we demonstrate that Munc13-4 plays a central role in the regulation of exocytosis of various sets of secretory organelles. However, mobilization of CD11b was not affected in Munc13-4-deficient neutrophils, indicating that secretory defects in these cells are limited to a selective group of exocytosable organelles.     

Cytotoxic T lymphocyte exocytosis: bring on the SNAREs!

Despite our general understanding of membrane traffic, the molecular machinery at the immunological synapse (IS) that regulates exocytosis of lytic granules from cytotoxic T lymphocytes (CTLs) remains elusive. The identification of disease-causing mutations in the small GTPase Rab27a, priming factor Munc13-4 and fusion protein syntaxin11 has defined an important role for these proteins in CTL exocytosis. In addition, the demonstration of a direct interaction in vitro between Rab27a and Munc13-4 suggests the possibility that the Rab27a-Munc13-4 cascade might regulate CTL exocytosis by engaging SNAREs such as syntaxin11. We propose that these SNAREs are likely to mediate the fusion of lytic granules with the plasma membrane of the IS.     

Munc13-4 Is a Rab11-binding Protein That Regulates Rab11-positive Vesicle Trafficking and Docking at the Plasma Membrane

The small GTPase Rab11 and its effectors control trafficking of recycling endosomes, receptor replenishment and the up-regulation of adhesion and adaptor molecules at the plasma membrane. Despite recent advances in the understanding of Rab11-regulated mechanisms, the final steps mediating docking and fusion of Rab11-positive vesicles at the plasma membrane are not fully understood. Munc13-4 is a docking factor proposed to regulate fusion through interactions with SNAREs. In hematopoietic cells, including neutrophils, Munc13-4 regulates exocytosis in a Rab27a-dependent manner, but its possible regulation of other GTPases has not been explored in detail. Here, we show that Munc13-4 binds to Rab11 and regulates the trafficking of Rab11-containing vesicles. Using a novel Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay, we demonstrate that Munc13-4 binds to Rab11a but not to dominant negative Rab11a. Immunoprecipitation analysis confirmed the specificity of the interaction between Munc13-4 and Rab11, and super-resolution microscopy studies support the interaction of endogenous Munc13-4 with Rab11 at the single molecule level in neutrophils. Vesicular dynamic analysis shows the common spatio-temporal distribution of Munc13-4 and Rab11, while expression of a calcium binding-deficient mutant of Munc13-4 significantly affected Rab11 trafficking. Munc13-4-deficient neutrophils showed normal endocytosis, but the trafficking, up-regulation, and retention of Rab11-positive vesicles at the plasma membrane was significantly impaired. This correlated with deficient NADPH oxidase activation at the plasma membrane in response to Rab11 interference. Our data demonstrate that Munc13-4 is a Rab11-binding partner that regulates the final steps of Rab11-positive vesicle docking at the plasma membrane.     

Rab27a negatively regulates CFTR chloride channel function in colonic epithelia: involvement of the effector proteins in the regulatory mechanism

Cystic fibrosis, an autosomal recessive disorder, is caused by the disruption of biosynthesis or function of CFTR. CFTR regulatory mechanisms include channel transport to plasma membrane and protein-protein interactions. Rab proteins are small GTPases involved in vesicle transport, docking, and fusion. The colorectal epithelial HT-29 cells natively express CFTR and respond to cAMP with an increase in CFTR-mediated currents. DPC-inhibited currents could be completely eliminated with CFTR-specific SiRNA. Over-expression of Rab27a inhibited, while isoform specific SiRNA and Rab27a antibody stimulated CFTR-mediated currents in HT-29 cells. CFTR activity is inhibited both by Rab27a (Q78L) (constitutive active GTP-bound form of Rab27a) and Rab27a (T23N) (constitutive negative form that mimics the GDP-bound form). Rab27a mediated effects could be reversed by Rab27a-binding proteins, the synaptotagmin-like protein (SLP-5) and Munc13-4 accessory protein (a putative priming factor for exocytosis). The SLP reversal of Rab27a effect was restricted to C2A/C2B domains while the SHD motif imparted little more inhibition. The CFTR-mediated currents remain unaffected by Rab3 though SLP-5 appears to weakly bind it. The immunoprecipitation experiments suggest protein-protein interactions between Rab27a and CFTR. Rab27a appears to impair CFTR appearance at the cell surface by trapping CFTR in the intracellular compartments. Munc13-4 and SLP-5, on the other hand, limit Rab27a availability to CFTR, thus minimizing its effect on channel function. These observations decisively prove that Rab27a is involved in CFTR channel regulation through protein-protein interactions involving Munc13-4 and SLP-5 effector proteins, and thus could be a potential target for cystic fibrosis therapy.     

Functional characterization of two RAB27A missense mutations found in Griscelli syndrome type 2

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.     

Munc13-4 restricts motility of Rab27a-expressing vesicles to facilitate lipopolysaccharide-induced priming of exocytosis in neutrophils

LPS is an efficient sensitizer of the neutrophil exocytic response to a second stimulus. Although neutrophil exocytosis in response to pathogen-derived molecules plays an important role in the innate immune response to infections, the molecular mechanism underlying LPS-dependent regulation of neutrophil exocytosis is currently unknown. The small GTPase Rab27a and its effector Munc13-4 regulate exocytosis in hematopoietic cells. Whether Rab27a and Munc13-4 modulate discrete steps or the same steps during exocytosis also remains unknown. Here, using Munc13-4- and Rab27a-deficient neutrophils, we analyzed the mechanism of lipopolysaccharide-dependent vesicular priming to amplify exocytosis of azurophilic granules. We found that both Munc13-4 and Rab27a are necessary to mediate LPS-dependent priming of exocytosis. However, we show that LPS-induced mobilization of a small population of readily releasable vesicles is a Munc13-4-dependent but Rab27a-independent process. LPS-induced priming regulation could not be fully explained by secretory organelle maturation as the redistribution of the secretory proteins Rab27a or Munc13-4 in response to LPS treatment was minimal. Using total internal reflection fluorescence microscopy and a novel mouse model expressing EGFP-Rab27a under the endogenous Rab27a promoter but lacking Munc13-4, we demonstrate that Munc13-4 is essential for the mechanism of LPS-dependent exocytosis in neutrophils and unraveled a novel mechanism of vesicular dynamics in which Munc13-4 restricts motility of Rab27a-expressing vesicles to facilitate lipopolysaccharide-induced priming of exocytosis.     

Expression of Rab3D N135I Inhibits Regulated Secretion of ACTH in AtT-20 Cells

Rab proteins are small molecular weight GTPases that control vesicular traffic in eucaryotic cells. A subset of Rab proteins, the Rab3 proteins are thought to play an important role in regulated exocytosis of vesicles. In transfected AtT-20 cells expressing wild-type Rab3D, we find that a fraction of the protein is associated with dense core granules. In the same cells, expression of a mutated isoform of Rab3D, Rab3D N135I, inhibits positioning of dense core granules near the plasma membrane, blocks regulated secretion of mature ACTH, and impairs association of Rab3A to membranes. Expression of Rab3D N135I does not change the levels of ACTH precursor or the efficiency with which the precursor is processed into ACTH hormone and packaged into dense core granules. We also find that cells expressing mutated Rab3D differentiate to the same extent as untransfected AtT-20 cells. We conclude that expression of Rab3D N135I specifically impairs late membrane trafficking events necessary for ACTH hormone secretion.     

EPI64B Acts as a GTPase-activating Protein for Rab27B in Pancreatic Acinar Cells

The small GTPase Rab27B localizes to the zymogen granule membranes and plays an important role in regulating protein secretion by pancreatic acinar cells, as does Rab3D. A common guanine nucleotide exchange factor (GEF) for Rab3 and Rab27 has been reported; however, the GTPase-activating protein (GAP) specific for Rab27B has not been identified. In this study, the expression in mouse pancreatic acini of two candidate Tre-2/Bub2/Cdc16 (TBC) domain-containing proteins, EPI64 (TBC1D10A) and EPI64B (TBC1D10B), was first demonstrated. Their GAP activity on digestive enzyme secretion was examined by adenovirus-mediated overexpression of EPI64 and EPI64B in isolated pancreatic acini. EPI64B almost completely abolished the GTP-bound form of Rab27B, without affecting GTP-Rab3D. Overexpression of EPI64B also enhanced amylase release. This enhanced release was independent of Rab27A, but dependent on Rab27B, as shown using acini from genetically modified mice. EPI64 had a mild effect on both GTP-Rab27B and amylase release. Co-overexpression of EPI64B with Rab27B can reverse the inhibitory effect of Rab27B on amylase release. Mutations that block the GAP activity decreased the inhibitory effect of EPI64B on the GTP-bound state of Rab27B and abolished the enhancing effect of EPI64B on the amylase release. These data suggest that EPI64B can serve as a potential physiological GAP for Rab27B and thereby participate in the regulation of exocytosis in pancreatic acinar cells.     

Munc13-4 is an effector of rab27a and controls secretion of lysosomes in hematopoietic cells

Griscelli syndrome type 2 (GS2) is a genetic disorder in which patients exhibit life-threatening defects of cytotoxic T lymphocytes (CTLs) whose lytic granules fail to dock on the plasma membrane and therefore do not release their contents. The disease is caused by the absence of functional rab27a, but how rab27a controls secretion of lytic granule contents remains elusive. Mutations in Munc13-4 cause familial hemophagocytic lymphohistiocytosis subtype 3 (FHL3), a disease phenotypically related to GS2. We show that Munc13-4 is a direct partner of rab27a. The two proteins are highly expressed in CTLs and mast cells where they colocalize on secretory lysosomes. The region comprising the Munc13 homology domains is essential for the localization of Munc13-4 to secretory lysosomes. The GS2 mutant rab27aW73G strongly reduced binding to Munc13-4, whereas the FHL3 mutant Munc13-4Delta608-611 failed to bind rab27a. Overexpression of Munc13-4 enhanced degranulation of secretory lysosomes in mast cells, showing that it has a positive regulatory role in secretory lysosome fusion. We suggest that the secretion defects seen in GS2 and FHL3 have a common origin, and we propose that the rab27a/Munc13-4 complex is an essential regulator of secretory granule fusion with the plasma membrane in hematopoietic cells. Mutations in either of the two genes prevent formation of this complex and abolish secretion.     

Slp4-a/granuphilin-a inhibits dense-core vesicle exocytosis through interaction with the GDP-bound form of Rab27A in PC12 cells

Slp4-a (synaptotagmin-like protein 4-a)/granuphilin-a is specifically localized on dense-core vesicles in PC12 cells and negatively controls dense-core vesicle exocytosis through specific interaction with Rab27A via the N-terminal Slp homology domain (SHD) (Fukuda, M., Kanno, E., Saegusa, C., Ogata, Y., and Kuroda, T. S. (2002) J. Biol. Chem. 277, 39673-39678). However, the mechanism of the inhibition by Slp4-a has never been elucidated at the molecular level and is still a matter of controversy. In this study, I discovered an unexpected biochemical property of Slp4-a, that Slp4-a, but not other Rab27 effectors reported thus far, is capable of interacting with both Rab27A(T23N), a dominant negative form that mimics the GDP-bound form, and Rab27A(Q78L), a dominant active form that mimics the GTP-bound form, whereas Slp4-a specifically recognizes the GTP-bound form of Rab3A and Rab8A and does not recognize their GDP-bound form. I show by deletion and mutation analyses that the TGDWFY sequence in SHD2 is essential for Rab27A(T23N) binding, whereas SHD1 is involved in Rab27A(Q78L) binding. I further show by immunoprecipitation and cotransfection assays that Munc18-1, but not syntaxin IA, directly interacts with the C-terminal domain of Slp4-a in a Rab27A-independent manner. Expression of Slp4-a mutants that lack Rab27A(T23N) binding activity (i.e. specific binding to Rab27A(Q78L)) completely reverses the inhibitory effect of the wild-type Slp4-a on high KCl-dependent neuropeptide Y secretion in PC12 cells. The results strongly indicate that interaction of Slp4-a with the GDP-bound form of Rab27A, not with syntaxin IA or Munc18-1, is the primary reason that Slp4-a expression inhibits dense core vesicle exocytosis in PC12 cells.     

Localization of the small monomeric GTPases Rab3D and Rab3A in the AtT-20 rat pituitary cell line

We investigated the cellular localization of the small GTPases Rab3D and Rab3A in AtT-20 cells treated with the drug Brefeldin A. Brefeldin A induces the redistribution of the Golgi complex into the endoplasmic reticulum and tubulation of endosomes. However, in Brefeldin A-treated wild-type AtT-20 cells, both Rab3D and Rab3A retained their distribution, indicating that they belong to a nonendosomal, post-Golgi compartment. Immunoelectron microscopy experiments indicated that both Rab3D and Rab3A localized to the ACTH-containing, large dense core granules. In contrast, in cell clones overexpressing a mutated form of Rab3D (Rab3D N135I), Rab3A did not localize to the dense core granules. Moreover, since our previous results showed that overexpression of Rab3D N135I severely impaired regulated ACTH secretion in AtT-20 cells, we sought to determine whether the impairment could depend on a redistribution of two key components of the regulated exocytosis machinery, synaptotagmin and SNAP-25. As far as synaptotagmin was concerned, in cell clones overexpressing Rab3D N135I, the protein did not localize close to the plasma membrane, in agreement with the previously reported defective docking of dense core granules to the plasma membrane. Immunofluorescence experiments showed that SNAP-25 did not change its localization in these cell clones. All in all, our findings strengthen the notion that both Rab3D and Rab3A are associated with the dense core granule compartment of AtT-20 cells, and that the impairment in the ACTH secretion caused by overexpression of a mutated Rab3D form is likely to be due to a lacking of granule docking to the plasma membrane, possibly because Rab3A fails to associate with the granules.     

Versatile role of Rab27 in membrane trafficking: focus on the Rab27 effector families

Rab27A was the only Rab protein whose dysfunction was found to cause human immunodeficiency. Since Griscelli syndrome patients (i.e., Rab27A-deficient) exhibit silvery hair color (i.e., pigmentary dilution) in addition to loss of cytotoxic killing activity by cytotoxic T lymphocytes, and Rab27A protein is expressed in a wide variety of secretory cells, Rab27A (or its closely related isoform Rab27B) has been implicated in the regulation of different types of membrane trafficking, including melanosome transport and various regulated secretion events. How does Rab27 protein regulate these different types of membrane trafficking? Recent discoveries of three different families of Rab27-binding proteins (a total of eleven distinct proteins) have supplied an important clue to the answer of this question: different types of Rab27 effectors function in different cell types. In this review I describe the literature on the identification of Rab27-binding proteins (i.e., the synaptotagmin-like protein (Slp) family with tandem C2 Ca(2+)-binding motifs, the Slac2 family without any C2 motifs, and Munc13-4, a putative priming factor for exocytosis) and the current state of our understanding of the molecular mechanism of the Rab27-dependent membrane trafficking.     

Rab27 Effectors,Pleiotropic Regulators in Secretory Pathways

Rab27, a member of the small GTPase Rab family, is widely conserved in metazoan, and two Rab27 isoforms, Rab27A and Rab27B, are present in vertebrates. Rab27A was the first Rab protein whose dysfunction was found to cause a human hereditary disease, type 2 Griscelli syndrome, which is characterized by silvery hair and immunodeficiency. The discovery in the 21st century of three distinct types of mammalian Rab27A effectors [synaptotagmin‐like protein (Slp), Slp homologue lacking C2 domains (Slac2), and Munc13‐4] that specifically bind active Rab27A has greatly accelerated our understanding not only of the molecular mechanisms of Rab27A‐mediated membrane traffic (e.g. melanosome transport and regulated secretion) but of the symptoms of Griscelli syndrome patients at the molecular level. Because Rab27B is widely expressed in various tissues together with Rab27A and has been found to have the ability to bind all of the Rab27A effectors that have been tested, Rab27A and Rab27B were initially thought to function redundantly by sharing common Rab27 effectors. However, recent evidence has indicated that by interacting with different Rab27 effectors Rab27A and Rab27B play different roles in special types of secretion (e.g. exosome secretion and mast cell secretion) even within the same cell type. In this review article, I describe the current state of our understanding of the functions of Rab27 effectors in secretory pathways .     

Genome-wide investigation of the Rab binding activity of RUN domains: development of a novel tool that specifically traps GTP-Rab35

The RUN domain is a less conserved protein motif that consists of approximately 70 amino acids, and because RUN domains are often found in proteins involved in the regulation of Rab small GTPases, the RUN domain has been suggested to be involved in Rab-mediated membrane trafficking, possibly as a Rab-binding site. However, since the Rab binding activity of most RUN domains has never been investigated, in this study we performed a genome-wide analysis of the Rab binding activity of the RUN domains of 19 different RUN domain-containing proteins by yeast two-hybrid assays with 60 different Rabs as bait. The results showed that only six of them interact with specific Rab isoforms with different Rab binding specificity, i.e., DENND5A/B with Rab6A/B, PLEKHM2 with Rab1A, RUFY2/3 with Rab33, and RUSC2 with Rab1/Rab35/Rab41. We also identified the minimal functional Rab35-binding site of RUSC2 (amino acid residues 982-1199) and succeeded in developing a novel GTP-Rab35-specific trapper, which we named RBD35 (Rab-binding domain specific for Rab35). Recombinant RBD35 was found to trap GTP-Rab35 specifically both in vitro and in PC12 cells, and overexpression of fluorescently tagged RBD35 in PC12 cells strongly inhibited nerve growth factor-dependent neurite outgrowth.     

Munc13-4 reconstitutes calcium-dependent SNARE-mediated membrane fusion

Munc13-4 is a widely expressed member of the CAPS/Munc13 protein family proposed to function in priming secretory granules for exocytosis. Munc13-4 contains N- and C-terminal C2 domains (C2A and C2B) predicted to bind Ca(2+), but Ca(2+)-dependent regulation of Munc13-4 activity has not been described. The C2 domains bracket a predicted SNARE-binding domain, but whether Munc13-4 interacts with SNARE proteins is unknown. We report that Munc13-4 bound Ca(2+) and restored Ca(2+)-dependent granule exocytosis to permeable cells (platelets, mast, and neuroendocrine cells) dependent on putative Ca(2+)-binding residues in C2A and C2B. Munc13-4 exhibited Ca(2+)-stimulated SNARE interactions dependent on C2A and Ca(2+)-dependent membrane binding dependent on C2B. In an apparent coupling of membrane and SNARE binding, Munc13-4 stimulated SNARE-dependent liposome fusion dependent on putative Ca(2+)-binding residues in both C2A and C2B domains. Munc13-4 is the first priming factor shown to promote Ca(2+)-dependent SNARE complex formation and SNARE-mediated liposome fusion. These properties of Munc13-4 suggest its function as a Ca(2+) sensor at rate-limiting priming steps in granule exocytosis.