Synaptotagmin-like Protein Research Articles

Terminal transport of lytic granules to the immune synapse is mediated by the kinesin-1/Slp3/Rab27a complex

AbstractCytotoxic T lymphocytes kill target cells via the polarized secretion of cytotoxic granules at the immune synapse. The lytic granules are initially recruited around the polarized microtubule-organizing center. In a dynein-dependent transport process, the granules move along microtubules toward the microtubule-organizing center in the minus-end direction. Here, we found that a kinesin-1–dependent process is required for terminal transport and secretion of polarized lytic granule to the immune synapse. We show that synaptotagmin-like protein 3 (Slp3) is an effector of Rab27a in cytotoxic T lymphocytes and interacts with kinesin-1 through the tetratricopeptide repeat of the kinesin-1 light chain. Inhibition of the Rab27a/Slp3/kinesin-1 transport complex impairs lytic granule secretion. Our data provide further molecular insights into the key functional and regulatory mechanisms underlying the terminal transport of cytotoxic granules and the latter's secretion at the immune synapse.

Molecular Interactions and Membrane Targeting of Granuphilin C2 Domains: A Preliminary Study

Membrane-targeting proteins are crucial components of many cellular signaling pathways, including the secretion of insulin. Granuphilin (also known as synaptotagmin-like protein 4) is essential for proper insulin secretion and acts most likely in docking the exocytic vesicles to the plasma membrane. However, the precise mechanisms of its membrane targeting and regulation remain uncertain. Granuphilin contains two C2 domains (C2A and C2B) involved in the process of docking to the lipid bilayer of the plasma membrane.

Bioinformatics of the TULIP domain superfamily

Proteins of the BPI (bactericidal/permeability-increasing protein)-like family contain either one or two tandem copies of a fold that usually provides a tubular cavity for the binding of lipids. Bioinformatic analyses show that, in addition to its known members, which include BPI, LBP [LPS (lipopolysaccharide)-binding protein)], CETP (cholesteryl ester-transfer protein), PLTP (phospholipid-transfer protein) and PLUNC (palate, lung and nasal epithelium clone) protein, this family also includes other, more divergent groups containing hypothetical proteins from fungi, nematodes and deep-branching unicellular eukaryotes. More distantly, BPI-like proteins are related to a family of arthropod proteins that includes hormone-binding proteins (Takeout-like; previously described to adopt a BPI-like fold), allergens and several groups of uncharacterized proteins. At even greater evolutionary distance, BPI-like proteins are homologous with the SMP (synaptotagmin-like, mitochondrial and lipid-binding protein) domains, which are found in proteins associated with eukaryotic membrane processes. In particular, SMP domain-containing proteins of yeast form the ERMES [ER (endoplasmic reticulum)-mitochondria encounter structure], required for efficient phospholipid exchange between these organelles. This suggests that SMP domains themselves bind lipids and mediate their exchange between heterologous membranes. The most distant group of homologues we detected consists of uncharacterized animal proteins annotated as TM (transmembrane) 24. We propose to group these families together into one superfamily that we term as the TULIP (tubular lipid-binding) domain superfamily.

Phosphoinositides and Other Glycerophospholipids Interact with Protein C Inhibitor (PCI) and Modify Its Activity towards Activated Protein C (APC).

Abstract 2122Poster Board II-99Protein C Inhibitor (PCI, SERPINA5, PAI3) is a non-specific, secreted serine protease inhibitor (serpin) which circulates at low levels (5μg/mL or 90nM) in blood plasma (review: Geiger 2007, Suzuki 2008). First described as an inhibitor of activated protein C (APC) an anticoagulant serine protease in human plasma, it has been shown that PCI inactivates a variety of other proteases and has a wide tissue distribution.Some compounds like glycosaminoglycans (e.g. heparin, heparan sulfate) and certain phospholipids can modify PCI activity. Recently it was shown that single-stranded DNA aptamers stimulates the inhibitory activity of PCI towards APC in a glycosaminoglycan-like fashion (Müller 2009).In 2007 Malleier et al. analyzed the interaction of PCI with phosphatidylserine (PS), oxidized PS (OxPS) and oxidized phosphatidylethanolamin (OxPE). PS, OxPS and OxPE bind to PCI and enhance the stimulation of APC-inhibition 130 to 190-fold. In addition, PE supports the internalization of PCI by cells, and internalized PCI promotes phagocytosis of bacteria (Baumgärtner et al. 2007). JFC1 (synaptotagmin-like protein 1) was identified by our group as a new intracellular interaction partner of PCI and colocalization of PCI and CSN6, a subunit of the COP9 signalosome could be observed in lymphocytes.Here, we analyzed the interaction of PCI with phosphatidic acid (PA), phosphatidylglycerol (PG), cardiolipin (CL), phosphoinositides and derived second messengers like inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). To identify lipid-regions which are important for PCI binding we were focusing on differences in fatty acid composition and headgroup phosphorylation. The binding was studied by native PAGE, protein overlay assays (dot blot analysis) or ELISA. The stimulation of PCI activity towards APC was analyzed in functional assays using a low molecular weight substrate (S-2366).IP3 and inositol-1,3,4,5-tetrakisphosphate (IP4) did neither interact with PCI nor stimulate its activity towards APC.PCI bound to saturated, unsaturated and oxidized PA. The oxidized form of 1-palmitoyl-2-arachidonoyl-phosphatidic acid (OxPAPA) exhibited lower binding to PCI, but higher stimulatory activity on APC inhibition, as compared to unoxidized PAPA. Saturated dipalmitoyl-PA did not modulate PCI activity.From all studied lipids 1-palmitoyl-2-arachidonoyl-PG (PAPG) had the strongest stimulatory effect on APC-inhibition similar to 0.1μM low molecular weight heparin. Oxidation of PAPG led to a slight decrease and saturation to a complete loss in stimulatory activity.Also tetra-oleoyl-CL bound to PCI with high affinity and had a similar effect as oxidized PAPG and OxPAPA.All mono- and diphosphorylated phosphoinositides as well as phosphatidylinositol-3,4,5-triphosphate (PI3,4,5P3) bound to PCI as judged from binding assays. A mobility shift of PCI antigen on native PAGE was observed when PCI was incubated with phosphatidylinositol-3,5-diphosphate and phosphatidylinositol-4,5-diphosphate (PI4,5P2).Therefore different phospholipids modulate the activity of PCI, but on the other hand PCI may as well effect lipid signaling. As PI4,5P2 plays an important role as substrate for PI3-kinase we will take a closer look at the effect of PCI on the PI3K/PTEN system and on the activation of AKT (PKB) by PDK1. We will also study the influence of PCI on the generation of IP3 and DAG and its possible role in calcium signaling and protein kinase activation.So far we conclude that phosphoinositides and other glycerophospholipids may function as additional intracellular interaction partners of PCI. Disclosures:No relevant conflicts of interest to declare.

Synaptotagmin-like protein 1 interacts with the GTPase-activating protein Rap1GAP2 and regulates dense granule secretion in platelets

AbstractThe small guanine-nucleotide–binding protein Rap1 plays a key role in platelet aggregation and hemostasis, and we recently identified Rap1GAP2 as the only GTPase-activating protein of Rap1 in platelets. In search of Rap1GAP2-associated proteins, we performed yeast-2-hybrid screening and found synaptotagmin-like protein 1 (Slp1) as a new binding partner. We confirmed the interaction of Rap1GAP2 and Slp1 in transfected COS-1 and HeLa cells and at endogenous level in human platelets. Mapping studies showed that Rap1GAP2 binds through amino acids T524-K525-X-T527 within its C-terminus to the C2A domain of Slp1. Slp1 contains a Rab27-binding domain, and we demonstrate that Rap1GAP2, Slp1, and Rab27 form a trimeric complex in transfected cells and in platelets. Purified Slp1 dose-dependently decreased dense granule secretion in streptolysin-O–permeabilized platelets stimulated with calcium or guanosine 5′-O-[gamma-thio] triphosphate. The isolated C2A domain of Slp1 had a stimulatory effect on granule secretion and reversed the inhibitory effect of full-length Slp1. Purified Rap1GAP2 augmented dense granule secretion of permeabilized platelets, whereas deletion of the Slp1-binding TKXT motif abolished the effect of Rap1GAP2. We conclude that Slp1 inhibits dense granule secretion in platelets and that Rap1GAP2 modulates secretion by binding to Slp1.

Dysferlin Deficiency Shows Compensatory Induction of Rab27A/Slp2a That May Contribute to Inflammatory Onset

Mutations in the dysferlin gene cause limb girdle muscular dystrophy 2B (LGMD2B) and Miyoshi myopathy. Dysferlin-deficient cells show abnormalities in vesicular traffic and membrane repair although onset of symptoms is not commonly seen until the late teenage years and is often associated with subacute onset and marked muscle inflammation. To identify molecular networks specific to dysferlin-deficient muscle that might explain disease pathogenesis, muscle mRNA profiles from 10 mutation-positive LGMD2B/MM patients were compared with a disease control [LGMD2I; (n = 9)], and normal muscle samples (n = 11). Query of inflammatory pathways suggested LGMD2B-specific increases in co-stimulatory signaling between dendritic cells and T cells (CD86, CD28, and CTLA4), associated with localized expression of both versican and tenascin. LGMD2B muscle also showed an increase in vesicular trafficking pathway proteins not normally observed in muscle (synaptotagmin-like protein Slp2a/SYTL2 and the small GTPase Rab27A). We propose that Rab27A/Slp2a expression in LGMD2B muscle provides a compensatory vesicular trafficking pathway that is able to repair membrane damage in the absence of dysferlin. However, this same pathway may release endocytotic vesicle contents, resulting in an inflammatory microenvironment. As dysferlin deficiency has been shown to enhance phagocytosis by macrophages, together with our findings of abnormal myofiber endocytosis pathways and dendritic-T cell activation markers, these results suggest a model of immune and inflammatory network over-stimulation that may explain the subacute inflammatory presentation.

Structural characterization of soluble E-Syt2

The protein family of membrane-anchored extended synaptotagmin-like proteins (E-Syts) was recently discovered in humans. E-Syt1 to 3 each contain at least one transmembrane domain and three or five C2 domains. To investigate the whole C2 area of murine E-Syt2, highly pure recombinant E-Syt2 (rE-Syt2) covering all three C2 domains was isolated. The structure of rE-Syt2 was studied by small-angle X-ray scattering (SAXS) providing a three-dimensional image of a protein with three C2 domains. Calcium binding of rE-Syt2 triggered structural rearrangements and initiated reversible multimerization of the protein in vitro. Quantitative analysis of the calcium binding revealed an apparent binding constant of 100 μM. This is the first structural study of a multi-C2 protein, presumably involved in Ca-dependent signalling events.

Review: Molecular mechanism of docking of dense-core vesicles to the plasma membrane in neuroendocrine cells

Almost every cell in our organism releases proteins and other biological substances using a fundamental cellular process known as constitutive exocytosis. In contrast, in exocrine and endocrine glands, the cells are inherent in an additional and more refined release mechanism directly tuned by extracellular signals. This process, referred to as regulated exocytosis, ensures the timely delivery of molecules such as peptide hormones and digestive enzymes to the exact moment-to-moment requirements of the organism. Recent studies of the final step of hormone secretion have been shown that the monomeric GTPase Rab27 subfamily and its cell type-or tissue-specific Rab27-binding protein(s) [also called Rab27 effector(s)] exist on the hormone-containing dense-core vesicle membrane and regulate the secretion of peptide hormones in endocrine cells. Synaptotagmin-like proteins (Slps) and rabphilin contain an N-terminal Rab27-binding domain and C-terminal tandem C2 domains, and some of the Rab27-binding proteins have recently been shown to promote docking of dense-core vesicles to the plasma membrane in a variety of secretory cells. We discuss here the evidence supporting a molecular mechanism for the way that the Rab27 and Rab27 effector complex controls the docking step in the trafficking of dense-core vesicles in neuroendocrine cells.

Slp1 and Slp2‐a Localize to the Plasma Membrane of CTL and Contribute to Secretion from the Immunological Synapse

Rab27a is required for polarized secretion of lysosomes from cytotoxic T lymphocytes (CTLs) at the immunological synapse. A series of Rab27a-interacting proteins have been identified; however, only Munc13-4 has been found to be expressed in CTL. In this study, we screened for expression of the synaptotagmin-like proteins (Slps): Slp1/JFC1, Slp2-a/exophilin4, Slp3-a, Slp4/granuphilin, Slp5 and rabphilin in CTL. We found that both Slp1 and Slp2-a are expressed in CTL. Isoforms of Slp2-a in CTL showed variation of the linker region but conserved the C2A and C2B and Slp homology (SHD) domains. Both Slp1 and Slp2-a interact with Rab27a in CTL, and Slp2-a, but not Slp1, is rapidly degraded when Rab27a is absent. Slp2-a contains PEST-like sequences within its linker region, which render it susceptible to degradation. Both Slp1 and Slp2-a localize predominantly to the plasma membrane of both human and mouse CTLs, and we show that Slp2-a can focus tightly at the immunological synapse formed with a target cell. Individual knockouts of either Slp2-a or Slp1 fail to impair CTL-mediated killing of targets; however, overexpression of a dominant-negative construct consisting of the SHD of Slp2-a, which is 56% identical to that of Slp1, reduces target cell death, suggesting that both Slp1 and Slp2-a contribute to secretory lysosome exocytosis from CTL. These results suggest that both Slp1 and Slp2-a may form part of a docking complex, capturing secretory lysosomes at the immunological synapse.

Identification of Secretory Granule Phosphatidylinositol 4,5-Bisphosphate-interacting Proteins Using an Affinity Pulldown Strategy

Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) synthesis is required for calcium-dependent exocytosis in neurosecretory cells. We developed a PtdIns(4,5)P2 bead pulldown strategy combined with subcellular fractionation to identify endogenous chromaffin granule proteins that interact with PtdIns(4,5)P2. We identified two synaptotagmin isoforms, synaptotagmins 1 and 7; spectrin; alpha-adaptin; and synaptotagmin-like protein 4 (granuphilin) by mass spectrometry and Western blotting. The interaction between synaptotagmin 7 and PtdIns(4,5)P2 and its functional relevance was investigated. The 45-kDa isoform of synaptotagmin 7 was found to be highly expressed in adrenal chromaffin cells compared with PC12 cells and to mainly localize to secretory granules by subcellular fractionation, immunoisolation, and immunocytochemistry. We demonstrated that synaptotagmin 7 binds PtdIns(4,5)P2 via the C2B domain in the absence of calcium and via both the C2A and C2B domains in the presence of calcium. We mutated the polylysine stretch in synaptotagmin 7 C2B and demonstrated that this mutant domain lacks the calcium-independent PtdIns(4,5)P2 binding. Synaptotagmin 7 C2B domain inhibited catecholamine release from digitonin-permeabilized chromaffin cells, and this inhibition was abrogated with the C2B polylysine mutant. These data indicate that synaptotagmin 7 C2B-effector interactions, which occur via the polylysine stretch, including calcium-independent PtdIns(4,5)P2 binding, are important for chromaffin granule exocytosis.

E-Syts, a family of membranous Ca 2+ -sensor proteins with multiple C 2 domains

C(2) domains are autonomously folded protein modules that generally act as Ca(2+)- and phospholipid-binding domains and/or as protein-protein interaction domains. We now report the primary structures and biochemical properties of a family of evolutionarily conserved mammalian proteins, referred to as E-Syts, for extended synaptotagmin-like proteins. E-Syts contain an N-terminal transmembrane region, a central juxtamembranous domain that is conserved from yeast to human, and five (E-Syt1) or three (E-Syt2 and E-Syt3) C-terminal C(2) domains. Only the first E-Syt C(2) domain, the C(2)A domain, includes the complete sequence motif that is required for Ca(2+) binding in C(2) domains. Recombinant protein fragments of E-Syt2 that include the first C(2) domain are capable of Ca(2+)-dependent phospholipid binding at micromolar concentrations of free Ca(2+), suggesting that E-Syts bind Ca(2+) through their first C(2) domain in a phospholipid complex. E-Syts are ubiquitously expressed, but enriched in brain. Expression of myc-tagged E-Syt proteins in transfected cells demonstrated localization to intracellular membranes for E-Syt1 and to plasma membranes for E-Syt2 and E-Syt3. Structure/function studies showed that the plasma-membrane localization of E-Syt2 and E-Syt3 was directed by their C-terminal C(2)C domains. This result reveals an unexpected mechanism by which the C(2)C domains of E-Syt2 and E-Syt3 functions as a targeting motif that localizes these proteins into the plasma membrane independent of their transmembrane region. Viewed together, our findings suggest that E-Syts function as Ca(2+)-regulated intrinsic membrane proteins with multiple C(2) domains, expanding the repertoire of such proteins to a fourth class beyond synaptotagmins, ferlins, and MCTPs (multiple C(2) domain and transmembrane region proteins).

Rab27a is a key component of the secretory machinery of azurophilic granules in granulocytes

Neutrophils kill micro-organisms using microbicidal products that they release into the phagosome or into the extracellular space. The secretory machinery utilized by neutrophils is poorly characterized. We show that the small GTPase Rab27a is an essential component of the secretory machinery of azurophilic granules in granulocytes. Rab27a-deficient mice have impaired secretion of MPO (myeloperoxidase) into the plasma in response to lipopolysaccharide. Cell fractionation analysis revealed that Rab27a and the Rab27a effector protein JFC1/Slp1 (synaptotagmin-like protein 1) are distributed principally in the low-density fraction containing a minor population of MPO-containing granules. By immunofluorescence microscopy, we detected Rab27a and JFC1/Slp1 in a minor subpopulation of MPO-containing granules. Interference with the JFC1/Slp1-Rab27a secretory machinery impaired secretion of MPO in permeabilized neutrophils. The expression of Rab27a was dramatically increased when promyelocytic HL-60 cells were differentiated into granulocytes but not when they were differentiated into monocytes. Down-regulation of Rab27a in HL-60 cells by RNA interference did not affect JFC1/Slp1 expression but significantly decreased the secretion of MPO. Neither Rab27a nor JFC1/Slp1 was integrated into the phagolysosome membrane during phagocytosis. Neutrophils from Rab27a-deficient mice efficiently phagocytose zymosan opsonized particles and deliver MPO to the phagosome. We conclude that Rab27a and JFC1/Slp1 permit MPO release into the surrounding milieu and constitute key components of the secretory machinery of azurophilic granules in granulocytes. Our results suggest that the granules implicated in cargo release towards the surrounding milieu are molecularly and mechanistically different from those involved in their release towards the phagolysosome.

Characterization of the Rab8-specific membrane traffic route linked to protrusion formation

Rab8 has a drastic effect on cell shape, but the membrane trafficking route it regulates is poorly defined. Here, we show that endogenous and ectopically expressed Rab8 is associated with macropinosomes generated at ruffling membrane domains. These macropinosomes fuse or transform into tubules that move toward the cell center, from where they are recycled back to the leading edge. The biogenesis of these tubules is dependent on actin and microtubular dynamics. Expression of dominant-negative Rab8 mutants or depletion of Rab8 by RNA interference inhibit protrusion formation, but promote cell-cell adhesion and actin stress fiber formation, whereas expression of the constitutively active Rab8-Q67L has the opposite effect. Rab8 localization overlaps with both Rab11 and Arf6, and is functionally linked to Arf6. We also demonstrate that Rab8 activity is needed for the transport of transferrin and the transferrin receptor to the pericentriolar region and to cell protrusions, and that Rab8 controls the traffic of cholera toxin B to the Golgi compartment. Finally, Rab8 colocalizes and binds specifically to a synaptotagmin-like protein (Slp1/JFC1), which is involved in controlling Rab8 membrane dynamics. We propose that Rab8 regulates a membrane-recycling pathway that mediates protrusion formation.

Rab27 and its effectors in secretory granule exocytosis: a novel docking machinery composed of a Rab27·effector complex

A small GTPase Rab27 is present on secretory granules in a wide variety of secretory cells and on melanosomes in melanocytes, and it is involved in controlling the trafficking of these organelles through interaction with a cell-type- or tissue-specific Rab27 effector(s). Slps (synaptotagmin-like proteins) and rabphilin contain an N-terminal Rab27-binding domain and C-terminal tandem C2 domains, and some of the Rab27-binding proteins have recently been shown to promote docking of Rab27-bound organelles to the plasma membrane. This mini-review presents a model for how the Rab27.effector complex controls the docking step in the trafficking of Rab27-bound organelles. Our results indicate that Slp2-a, Slp4-a/granuphilin-a and rabphilin are capable of interacting with the plasma membrane directly or indirectly, and thus that these Rab27 effectors form a bridge between Rab27-bound organelles and the plasma membrane.

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.

Spatial control of actin organization at adherens junctions by a synaptotagmin-like protein

Epithelial tissues maintain a robust architecture during development. This fundamental property relies on intercellular adhesion through the formation of adherens junctions containing E-cadherin molecules. Localization of E-cadherin is stabilized through a pathway involving the recruitment of actin filaments by E-cadherin. Here we identify an additional pathway that organizes actin filaments in the apical junctional region (AJR) where adherens junctions form in embryonic epithelia. This pathway is controlled by Bitesize (Btsz), a synaptotagmin-like protein that is recruited in the AJR independently of E-cadherin and is required for epithelial stability in Drosophila embryos. On loss of btsz, E-cadherin is recruited normally to the AJR, but is not stabilized properly and actin filaments fail to form a stable continuous network. In the absence of E-cadherin, actin filaments are stable for a longer time than they are in btsz mutants. We identify two polarized cues that localize Btsz: phosphatidylinositol (4,5)-bisphosphate, to which Btsz binds; and Par-3. We show that Btsz binds to the Ezrin-Radixin-Moesin protein Moesin, an F-actin-binding protein that is localized apically and is recruited in the AJR in a btsz-dependent manner. Expression of a dominant-negative form of Ezrin that does not bind F-actin phenocopies the loss of btsz. Thus, our data indicate that, through their interaction, Btsz and Moesin may mediate the proper organization of actin in a local domain, which in turn stabilizes E-cadherin. These results provide a mechanism for the spatial order of actin organization underlying junction stabilization in primary embryonic epithelia.

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.

Decreased basal mucus secretion by Slp2‐a‐deficient gastric surface mucous cells

Synaptotagmin-like protein (Slp) 2-a is a putative Rab27A/B-effector protein and is implicated in intracellular membrane transport. However, the precise tissue distribution of Slp2-a protein and its functions remain largely unknown. In this study we used a specific anti-Slp2-a antibody to investigate the tissue distribution of Slp2-a in mice and found that Slp2-a is most abundantly expressed in mouse stomach. Co-immunoprecipitation experiments indicated that Slp2-a interacts with Rab27A/B in vivo. We also discovered that Slp2-a and Rab27A/B are predominantly localized at the apical region of gastric-surface mucous cells, where mucus granules are accumulated. Analysis of Slp2-a mutant mice generated by homologous recombination showed a reduced number of mucus granules, a deficiency of granule docking with the apical plasma membrane in the gastric-surface mucous cells and reduction of mucus secretion by Slp2-a-deficient gastric primary cells. Based on these results, we propose that Slp2-a is part of the mucin secretory machinery in surface mucous cells of mouse stomach.

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.

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.