Granuphilin molecularly docks insulin granules to the fusion machinery

The Rab27a effector granuphilin is specifically localized on insulin granules and is involved in their exocytosis. Here we show that the number of insulin granules morphologically docked to the plasma membrane is markedly reduced in granuphilin-deficient beta cells. Surprisingly, despite the docking defect, the exocytosis of insulin granules in response to a physiological glucose stimulus is significantly augmented, which results in increased glucose tolerance in granuphilin-null mice. The enhanced secretion in mutant beta cells is correlated with a decrease in the formation of the fusion-incompetent syntaxin-1a-Munc18-1 complex, with which granuphilin normally interacts. Furthermore, in contrast to wild-type granuphilin, its mutant that is defective in binding to syntaxin-1a fails to restore granule docking or the protein level of syntaxin-1a in granuphilin-null beta cells. Thus, granuphilin not only is essential for the docking of insulin granules but simultaneously imposes a fusion constraint on them through an interaction with the syntaxin-1a fusion machinery. These findings provide a novel paradigm for the docking machinery in regulated exocytosis.     

Docking is not a prerequisite but a temporal constraint for fusion of secretory granules

We examined secretory granule dynamics using total internal reflection fluorescence microscopy in normal pancreatic β cells and their mutants devoid of Rab27a and/or its effector, granuphilin, which play critical roles in the docking and recruitment of insulin granules to the plasma membrane. In the early phase of glucose stimulation in wild-type cells, we observed marked fusion of granules recruited from a relatively distant area, in parallel with that from granules located underneath the plasma membrane. Furthermore, despite a lack of granules directly attached to the plasma membrane, both spontaneous and evoked fusion was increased in granuphilin-null cells. In addition to these granuphilin-null phenotypes, Rab27a/granuphilin doubly deficient cells showed the decreases in granules located next to the docked area and in fusion from granules near the plasma membrane in the early phase of glucose-stimulated secretion, similar to Rab27a-mutated cells. Thus, the two proteins play nonoverlapping roles in insulin exocytosis: granuphilin acts on the granules underneath the plasma membrane, whereas Rab27a acts on those in a more distal area. These findings demonstrate that, in contrast to our conventional understanding, stable attachment of secretory granules to the plasma membrane is not prerequisite but temporally inhibitory for both spontaneous and evoked fusion.     

Exophilin4/Slp2-a targets glucagon granules to the plasma membrane through unique Ca2+-inhibitory phospholipid-binding activity of the C2A domain

Rab27a and Rab27b have recently been recognized to play versatile roles in regulating the exocytosis of secretory granules and lysosome-related organelles by using multiple effector proteins. However, the precise roles of these effector proteins in particular cell types largely remain uncharacterized, except for those in pancreatic beta cells and in melanocytes. Here, we showed that one of the Rab27a/b effectors, exophilin4/Slp2-a, is specifically expressed in pancreatic alpha cells, in contrast to another effector, granuphilin, in beta cells. Like granuphilin toward insulin granules, exophilin4 promotes the targeting of glucagon granules to the plasma membrane. Although the interaction of granuphilin with syntaxin-1a is critical for the targeting activity, exophilin4 does this primarily through the affinity of its C2A domain toward the plasma membrane phospholipids phosphatidylserine and phosphatidylinositol-4,5-bisphosphate. Notably, the binding activity to phosphatidylserine is inhibited by a physiological range of the Ca(2+) concentration attained after secretagogue stimulation, which presents a striking contrast to the Ca(2+)-stimulatory activity of the C2A domain of synaptotagmin I. Analyses of the mutant suggested that this novel Ca(2+)-inhibitory phospholipid-binding activity not only mediates docking but also modulates the subsequent fusion of the secretory granules.     

Munc 18-1 and granuphilin collaborate during insulin granule exocytosis

Munc 18-1 is a member of the Sec/Munc family of syntaxin-binding proteins known to bind to the plasma membrane Q-SNARE syntaxin1 and whose precise role in regulated exocytosis remains controversial. Here, we show that Munc 18-1 plays a positive role in regulated insulin secretion from pancreatic beta cells. Munc 18-1 depletion caused a loss in the secretory capacity of both transiently transfected INS 1E cells and a stable clone with tetracycline-regulated Munc 18-1 RNA interference. In addition, Munc 18-1-depleted cells exhibited defective docking of insulin granules to the plasma membrane and accumulated insulin in the trans Golgi network. Furthermore, glucose stimulation after Munc 18-1 depletion resulted in the rapid formation of autophagosomes. In contrast, overexpression of Munc 18-1 had no effect on insulin secretion. Although there was no detectable interaction between Munc 18-1 and Munc-18-interacting protein 1 or calcium/calmodulin-dependent serine protein kinase, Munc 18-1 associated with the granular protein granuphilin. This association was regulated by glucose and was required for the specific interaction of insulin granules with syntaxin1. We conclude that Munc 18-1 and granuphilin collaborate in the docking of insulin granules to the plasma membrane in an initial fusion-incompetent state, with Munc 18-1 subsequently playing a positive role in a later stage of insulin granule exocytosis.     

Rab27b is expressed in a wide range of exocytic cells and involved in the delivery of secretory granules near the plasma membrane

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.     

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.     

The Slp4-a linker domain controls exocytosis through interaction with Munc18-1.syntaxin-1a complex

Synaptotagmin-like protein 4-a (Slp4-a)/granuphilin-a is specifically localized on dense-core vesicles in certain neuroendocrine cells and negatively controls dense-core vesicle exocytosis through specific interaction with Rab27A. However, the precise molecular mechanism of its inhibitory effect on exocytosis has never been elucidated and is still a matter of controversy. Here we show by deletion and chimeric analyses that the linker domain of Slp4-a interacts with the Munc18-1.syntaxin-1a complex by directly binding to Munc18-1 and that this interaction promotes docking of dense-core vesicles to the plasma membrane in PC12 cells. Despite increasing the number of plasma membrane docked vesicles, expression of Slp4-a strongly inhibited high-KCl-induced dense-core vesicle exocytosis. The inhibitory effect by Slp4-a is absolutely dependent on the linker domain of Slp4-a, because substitution of the linker domain of Slp4-a by that of Slp5 (the closest isoform of Slp4-a that cannot bind the Munc18-1.syntaxin-1a complex) completely abrogated the inhibitory effect. Our findings reveal a novel docking machinery for dense-core vesicle exocytosis: Slp4-a simultaneously interacts with Rab27A and Munc18-1 on the dense-core vesicle and with syntaxin-1a in the plasma membrane.     

Munc18-1 regulates first-phase insulin release by promoting granule docking to multiple syntaxin isoforms

Attenuated levels of the Sec1/Munc18 (SM) protein Munc18-1 in human islet β-cells is coincident with type 2 diabetes, although how Munc18-1 facilitates insulin secretion remains enigmatic. Herein, using conventional Munc18-1(+/-) and β-cell specific Munc18-1(-/-) knock-out mice, we establish that Munc18-1 is required for the first phase of insulin secretion. Conversely, human islets expressing elevated levels of Munc18-1 elicited significant potentiation of only first-phase insulin release. Insulin secretory changes positively correlated with insulin granule number at the plasma membrane: Munc18-1-deficient cells lacked 35% of the normal component of pre-docked insulin secretory granules, whereas cells with elevated levels of Munc18-1 exhibited a ～20% increase in pre-docked granule number. Pre-docked syntaxin 1-based SNARE complexes bound by Munc18-1 were detected in β-cell lysates but, surprisingly, were reduced by elevation of Munc18-1 levels. Paradoxically, elevated Munc18-1 levels coincided with increased binding of syntaxin 4 to VAMP2 at the plasma membrane. Accordingly, syntaxin 4 was a requisite for Munc18-1 potentiation of insulin release. Munc18c, the cognate SM isoform for syntaxin 4, failed to bind SNARE complexes. Given that Munc18-1 does not pair with syntaxin 4, these data suggest a novel indirect role for Munc18-1 in facilitating syntaxin 4-mediated granule pre-docking to support first-phase insulin exocytosis.     

Rab27 effector granuphilin promotes the plasma membrane targeting of insulin granules via interaction with syntaxin 1a

Secretory vesicle exocytosis is a highly regulated process involving vesicle targeting, priming, and membrane fusion. Rabs and SNAREs play a central role in executing these processes. We have shown recently that Rab27a and its effector, granuphilin, are involved in the exocytosis of insulin-containing secretory granules through a direct interaction with the plasma membrane syntaxin 1a in pancreatic beta cells. Here, we demonstrate that fluorescence-labeled insulin granules are peripherally accumulated in cells overexpressing granuphilin. The peripheral location of granules is well overlapped with both localizations of granuphilin and syntaxin 1a. The plasma membrane targeting of secretory granules is promoted by wild-type granuphilin but not by granuphilin mutants that are defective in binding to either Rab27a or syntaxin 1a. Granuphilin directly binds to the H3 domain of syntaxin 1a containing its SNARE motif. Moreover, introduction of the H3 domain into beta cells induces a dissociation of the native granuphilin-syntaxin complex and a marked reduction of newly docked granules. These results indicate that granuphilin plays a role in tethering insulin granules to the plasma membrane by an interaction with both Rab27a and syntaxin 1a. The complex formation of these three proteins may contribute to the specificity of the targeting process during the exocytosis of insulin granules.     

Granuphilin modulates the exocytosis of secretory granules through interaction with syntaxin 1a

The molecular mechanism for the regulated exocytosis of dense-core granules in endocrine cells remains relatively uncharacterized compared to that of synaptic vesicles in neurons. A novel set of Rab and its effector, Rab27a/granuphilin, which is localized on insulin granules in pancreatic beta cells, was recently identified. Here we demonstrate that granuphilin directly binds to syntaxin 1a on the plasma membrane, and this interaction is regulated by Rab27a. Granuphilin shows affinity to syntaxin 1a with a closed conformation but not to mutant syntaxin 1a, which adopts an open conformation constitutively. Overexpression of granuphilin significantly enhances basal insulin secretion but profoundly inhibits high K(+)-induced insulin secretion. The effect of granuphilin on insulin secretion was impaired by its mutation that disrupts the binding to either Rab27a or syntaxin 1a. Thus, granuphilin is the first regulator in the exocytotic pathway that functions by directly connecting two critical vesicle transport proteins, Rab and SNARE.     

A gain-of-function mutant of Munc18-1 stimulates secretory granule recruitment and exocytosis and reveals a direct interaction of Munc18-1 with Rab3

Munc18-1 plays a crucial role in regulated exocytosis in neurons and neuroendocrine cells through modulation of vesicle docking and membrane fusion. The molecular basis for Munc18 function is still unclear, as are the links with Rabs and SNARE [SNAP (soluble N-ethylmaleimide-sensitive factor-attachment protein) receptor] proteins that are also required. Munc18-1 can bind to SNAREs through at least three modes of interaction, including binding to the closed conformation of syntaxin 1. Using a gain-of-function mutant of Munc18-1 (E466K), which is based on a mutation in the related yeast protein Sly1p, we have identified a direct interaction of Munc18-1 with Rab3A, which is increased by the mutation. Expression of Munc18-1 with the E466K mutation increased exocytosis in adrenal chromaffin cells and PC12 cells (pheochromocytoma cells) and was found to increase the density of secretory granules at the periphery of PC12 cells, suggesting a stimulatory effect on granule recruitment through docking or tethering. Both the increase in exocytosis and changes in granule distribution appear to require Munc18-1 E466K binding to the closed form of syntaxin 1, suggesting a role for this interaction in bridging Rab- and SNARE-mediated events in exocytosis.     

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.     

The role of Rab3a in secretory vesicle docking requires association/dissociation of guanidine phosphates and Munc18-1

Rab3a is a small GTPase that binds selectively to secretory vesicles and switches between active, GTP-bound and inactive, GDP-bound conformations. In yeast, Rab and SM-genes interact genetically to promote vesicle targeting/fusion. We tested different Rab3a conformations and genetic interactions with the SM-gene munc18-1 on the docking function of Rab3a in mammalian chromaffin cells. We expressed Rab3a mutants locked in the GTP- or GDP-bound form in wild-type and munc18-1 null mutant cells and analyzed secretory vesicle distribution. We confirmed that wild-type Rab3a promotes vesicle docking in wild-type cells. Unexpectedly, both GTP- and GDP-locked Rab3a mutants did not promote docking. Furthermore, wild-type Rab3a did not promote docking in munc18-1 null cells and GTP- and GDP-Rab3a both decreased the amount of docked vesicles. The results show that GTP- and GDP-locked conformations do not support a Munc18-1 dependent role of Rab3a in docking. This suggests that nucleotide cycling is required to support docking and that this action of Rab3a is upstream of Munc18-1.     

Slp4-a/granuphilin-a interacts with syntaxin-2/3 in a Munc18-2-dependent manner

Slp4-a/granuphilin-a was originally described as a protein specifically associated with insulin-containing granules in pancreatic beta-cells, but it was subsequently found to be present on amylase-containing granules in parotid acinar cells. Although Slp4-a has been suggested to control insulin secretion through interaction with syntaxin-1a and/or Munc18-1, nothing is known about the binding partner(s) of Slp4-a during amylase release from parotid acinar cells, which do not endogenously express either syntaxin-1a or Munc18-1. In this study we systematically investigated the interaction between syntaxin-1-5 and Munc18-1-3 by co-immunoprecipitation assay using COS-7 cells and discovered that Slp4-a interacts with a closed conformation of syntaxin-2/3 in a Munc18-2-dependent manner, whereas Munc18-2 itself hardly interacts with Slp4-a at all. By contrast, Slp4-a was found to strongly interact with Munc18-1 regardless of the presence of syntaxin-2/3, and syntaxin-2/3 co-immunoprecipitated with Slp4-a only in the presence of Munc18-1/2. Deletion analysis showed that the syntaxin-2/3 (or Munc18-1/2)-binding site is a linker domain of Slp4-a (amino acid residues 144-354), a previously uncharacterized region located between the N-terminal Rab27A binding domain and the C2A domain. We also found that the Slp4-a.syntaxin-2 complex is actually present in rat parotid glands and that introduction of the antibody against Slp4-a linker domain into streptolysin O-permeabilized parotid acinar cells severely attenuates isoproterenol-stimulated amylase release, possibly by disrupting the interaction between Slp4-a and syntaxin-2/3 (or Munc18-2). These results suggest that Slp4-a modulates amylase release from parotid acinar cells through interaction with syntaxin-2/3 on the apical plasma membrane.     

Exophilin8 transiently clusters insulin granules at the actin-rich cell cortex prior to exocytosis

Exophilin8/MyRIP/Slac2-c is an effector protein of the small GTPase Rab27a and is specifically localized on retinal melanosomes and secretory granules. We investigated the role of exophilin8 in insulin granule trafficking. Exogenous expression of exophilin8 in pancreatic β cells or their cell line, MIN6, polarized (exophilin8-positive) insulin granules at the cell corners, where both cortical actin and the microtubule plus-end-binding protein, EB1, were present. Mutation analyses indicated that the ability of exophilin8 to act as a linker between Rab27a and myosin Va is essential for its granule-clustering activity. Moreover, exophilin8 and exophilin8-associated insulin granules were markedly stable and immobile. Total internal reflection fluorescence microscopy indicated that exophilin8 restricts the motion of insulin granules at a region deeper than that where another Rab27a effector, granuphilin, accumulates docked granules directly attached to the plasma membrane. However, the exophilin8-induced immobility of insulin granules was eliminated upon secretagogue stimulation and did not inhibit evoked exocytosis. Furthermore, exophilin8 depletion prevents insulin granules from being transported close to the plasma membrane and inhibits their fusion. These findings indicate that exophilin8 transiently traps insulin granules into the cortical actin network close to the microtubule plus-ends and supplies them for release during the stimulation.     

The roles of Rab27 and its effectors in the regulated secretory pathways

Regulated secretory pathways are highly developed in multicellular organisms as a means of intercellular communication. Each of these pathways harbors unique store organelles, such as granules in endocrine and exocrine tissues and melanosomes in melanocytes. It has recently been shown that the monomeric GTPase Rab27 subfamily regulates the exocytosis of these cell-specific store organelles. Furthermore, genetic alterations of Rab27a cause Griscelli syndrome in humans that manifests as pigmentary dilution of the skin and the hair and variable immunodeficiency due to defects in the transport of melanosomes in melanocytes and lytic granules in cytotoxic T-lymphocytes. Rab27 acts through organelle-specific effector proteins, such as granuphilin in pancreatic beta cells and melanophilin in melanocytes. The Rab27 and effector complex then interacts with proteins that are essential for membrane transport and fusion, such as syntaxin 1a and Munc18-1 for granuphilin and myosin Va for melanophilin. Genome information suggests that other putative Rab27 effector proteins, tentatively termed as exophilins or Slp/Slac2, are predicted to exist because these proteins share the conserved N-terminal Rab27-binding domain and show Rab27-binding activity in vitro or when overexpressed in cell lines. These findings suggest that the Rab27 subfamily regulates various exocytotic pathways using multiple organelle-specific effector proteins.     

Rab3A and Rab3D control the total granule number and the fraction of granules docked at the plasma membrane in PC12 cells

Rab proteins are Ras-like GTPases that regulate traffic along the secretory or endocytic pathways. Within the Rab family, Rab3 proteins are expressed at high levels in neurons and endocrine cells where they regulate release of dense core granules and synaptic vesicles. Immuno-electron microscopy shows that Rab3A and Rab3D can coexist on the same granule before and after docking. Using electron microscopy of transfected PC12 cells, we report that expression of wild-type Rab3A (or Rab3D) increases the total number of granules and the percentage that is docked at the plasma membrane. Mutated Rab3A N135I (or Rab3D N135I) decreases the total granule number and the fraction of granules docked to the plasma membrane. These data show that at least one of the functions of Rab3A and Rab3D proteins is to control the number of granules docked at the plasma membrane.     

The Rab27a/granuphilin complex regulates the exocytosis of insulin-containing dense-core granules

Recently, we identified and characterized a novel protein, granuphilin, whose domain structure is similar to that of the Rab3 effector protein rabphilin3 (J. Wang, T. Takeuchi, H. Yokota, and T. Izumi, J. Biol. Chem. 274:28542-28548, 1999). Screening its possible Rab partner by a yeast two-hybrid system revealed that an amino-terminal zinc-finger domain of granuphilin interacts with Rab27a. Granuphilin preferentially bound to the GTP form of Rab27a. Formation of the Rab27a/granuphilin complex was readily detected in the pancreatic beta cell line MIN6. Moreover, the tissue distributions of Rab27a and granuphilin are remarkably similar: both had significant and specific expression in pancreatic islets and in pituitary tissue, but no expression was noted in the brain. Analyses by immunofluorescence, immunoelectron microscopy, and sucrose density gradient subcellular fractionation showed that Rab27a and granuphilin are localized on the membrane of insulin granules. These findings suggest that granuphilin functions as a Rab27a effector protein in beta cells. Overexpression of wild-type Rab27a and its GTPase-deficient mutant significantly enhanced high K(+)-induced insulin secretion without affecting basal insulin release. Although Rab3a, another exocytotic Rab protein, has some similarities with Rab27a in primary sequence, intracellular distribution, and affinity toward granuphilin, overexpression of Rab3a caused different effects on insulin secretion. These results indicate that Rab27a is involved in the regulated exocytosis of conventional dense-core granules possibly through the interaction with granuphilin, in addition to its recently identified role in lysosome-related organelles.     

VAMP8-dependent fusion of recycling endosomes with the plasma membrane facilitates T lymphocyte cytotoxicity

Cytotoxic T lymphocytes (CTLs) eliminate infected and neoplastic cells through directed release of cytotoxic granule contents. Although multiple SNARE proteins have been implicated in cytotoxic granule exocytosis, the role of vesicular SNARE proteins, i.e., vesicle-associated membrane proteins (VAMPs), remains enigmatic. VAMP8 was posited to represent the cytotoxic granule vesicular SNARE protein mediating exocytosis in mice. In primary human CTLs, however, VAMP8 colocalized with Rab11a-positive recycling endosomes. Upon stimulation, these endosomes rapidly trafficked to and fused with the plasma membrane, preceding fusion of cytotoxic granules. Knockdown of VAMP8 blocked both recycling endosome and cytotoxic granule fusion at immune synapses, without affecting activating signaling. Mechanistically, VAMP8-dependent recycling endosomes deposited syntaxin-11 at immune synapses, facilitating assembly of plasma membrane SNARE complexes for cytotoxic granule fusion. Hence, cytotoxic granule exocytosis is a sequential, multivesicle fusion process requiring VAMP8-mediated recycling endosome fusion before cytotoxic granule fusion. Our findings imply that secretory granule exocytosis pathways in other cell types may also be more complex than previously appreciated.