Binding of Ca2+-independent C2 domains to lipid membranes: A multi-scale molecular dynamics study.

C2 domains facilitate protein interactions with lipid bilayers in either a Ca2+-dependent or -independent manner. We used molecular dynamics (MD) simulations to explore six Ca2+-independent C2 domains, from KIBRA, PI3KC2α, RIM2, PTEN, SHIP2, and Smurf2. In coarse-grained MD simulations these C2 domains formed transient interactions with zwitterionic bilayers, compared with longer-lived interactions with anionic bilayers containing phosphatidylinositol bisphosphate (PIP2). Type I C2 domains bound non-canonically via the front, back, or side of the ß sandwich, whereas type II C2 domains bound canonically, via the top loops. C2 domains interacted strongly with membranes containing PIP2, causing bound anionic lipids to cluster around the protein. Binding modes were refined via atomistic simulations. For PTEN and SHIP2, CG simulations of their phosphatase plus C2 domains with PIP2-containing bilayers were also performed, and the roles of the two domains in membrane localization compared. These studies establish a simulation protocol for membrane-recognition proteins.

RIM-binding protein couples synaptic vesicle recruitment to release sites.

At presynaptic active zones, arrays of large conserved scaffold proteins mediate fast and temporally precise release of synaptic vesicles (SVs). SV release sites could be identified by clusters of Munc13, which allow SVs to dock in defined nanoscale relation to Ca2+ channels. We here show in Drosophila that RIM-binding protein (RIM-BP) connects release sites physically and functionally to the ELKS family Bruchpilot (BRP)-based scaffold engaged in SV recruitment. The RIM-BP N-terminal domain, while dispensable for SV release site organization, was crucial for proper nanoscale patterning of the BRP scaffold and needed for SV recruitment of SVs under strong stimulation. Structural analysis further showed that the RIM-BP fibronectin domains form a "hinge" in the protein center, while the C-terminal SH3 domain tandem binds RIM, Munc13, and Ca2+ channels release machinery collectively. RIM-BPs' conserved domain architecture seemingly provides a relay to guide SVs from membrane far scaffolds into membrane close release sites.

Mutational Analysis of Rab3 Function for Controlling Active Zone Protein Composition at the Drosophila Neuromuscular Junction.

At synapses, the release of neurotransmitter is regulated by molecular machinery that aggregates at specialized presynaptic release sites termed active zones. The complement of active zone proteins at each site is a determinant of release efficacy and can be remodeled to alter synapse function. The small GTPase Rab3 was previously identified as playing a novel role that controls the distribution of active zone proteins to individual release sites at the Drosophila neuromuscular junction. Rab3 has been extensively studied for its role in the synaptic vesicle cycle; however, the mechanism by which Rab3 controls active zone development remains unknown. To explore this mechanism, we conducted a mutational analysis to determine the molecular and structural requirements of Rab3 function at Drosophila synapses. We find that GTP-binding is required for Rab3 to traffick to synapses and distribute active zone components across release sites. Conversely, the hydrolytic activity of Rab3 is unnecessary for this function. Through a structure-function analysis we identify specific residues within the effector-binding switch regions that are required for Rab3 function and determine that membrane attachment is essential. Our findings suggest that Rab3 controls the distribution of active zone components via a vesicle docking mechanism that is consistent with standard Rab protein function.

DYNLT (Tctex-1) forms a tripartite complex with dynein intermediate chain and RagA, hence linking this small GTPase to the dynein motor.

It has been suggested that DYNLT, a dynein light chain known to bind to various cellular and viral proteins, can function as a microtubule-cargo adaptor. Recent data showed that DYNLT links the small GTPase Rab3D to microtubules and, for this to occur, the DYNLT homodimer needs to display a binding site for dynein intermediate chain together with a binding site for the small GTPase. We have analysed in detail how RagA, another small GTPase, associates to DYNLT. After narrowing down the binding site of RagA to DYNLT we could identify that a ß strand, part of the RagA G3 box involved in nucleotide binding, mediates this association. Interestingly, we show that both microtubule-associated DYNLT and cytoplasmic DYNLT are equally able to bind to the small GTPases Rab3D and RagA. Using NMR spectroscopy, we analysed the binding of dynein intermediate chain and RagA to mammalian DYNLT. Our experiments identify residues of DYNLT affected by dynein intermediate chain binding and residues affected by RagA binding, hence distinguishing the docking site for each of them. In summary, our results shed light on the mechanisms adopted by DYNLT when binding to protein cargoes that become transported alongside microtubules bound to the dynein motor.

Structures of Drosophila melanogaster Rab2 and Rab3 bound to GMPPNP.

Rab GTPases belong to the large family of Ras proteins. They act as key regulators of membrane organization and intracellular trafficking. Functionally, they act as switches. In the active GTP-bound form they can bind to effector proteins to facilitate the delivery of transport vesicles. Upon stimulation, the GTP is hydrolyzed and the Rab proteins undergo conformational changes in their switch regions. This study focuses on Rab2 and Rab3 from Drosophila melanogaster. Whereas Rab2 is involved in vesicle transport between the Golgi and the endoplasmatic reticulum, Rab3 is a key player in exocytosis, and in the synapse it is involved in the assembly of the presynaptic active zone. Here, high-resolution crystal structures of Rab2 and Rab3 in complex with GMPPNP and Mg2+ are presented. In the structure of Rab3 a modified cysteine residue is observed with an enigmatic electron density attached to its thiol function.

Mutation spectrum in RAB3GAP1, RAB3GAP2, and RAB18 and genotype-phenotype correlations in warburg micro syndrome and Martsolf syndrome.

Warburg Micro syndrome and Martsolf syndrome (MS) are heterogeneous autosomal-recessive developmental disorders characterized by brain, eye, and endocrine abnormalities. Causative biallelic germline mutations have been identified in RAB3GAP1, RAB3GAP2, or RAB18, each of which encode proteins involved in membrane trafficking. This report provides an up to date overview of all known disease variants identified in 29 previously published families and 52 new families. One-hundred and forty-four Micro and nine Martsolf families were investigated, identifying mutations in RAB3GAP1 in 41% of cases, mutations in RAB3GAP2 in 7% of cases, and mutations in RAB18 in 5% of cases. These are listed in Leiden Open source Variation Databases, which was created by us for all three genes. Genotype-phenotype correlations for these genes have now established that the clinical phenotypes in Micro syndrome and MS represent a phenotypic continuum related to the nature and severity of the mutations present in the disease genes, with more deleterious mutations causing Micro syndrome and milder mutations causing MS. RAB18 has not yet been linked to the RAB3 pathways, but mutations in all three genes cause an indistinguishable phenotype, making it likely that there is some overlap. There is considerable genetic heterogeneity for these disorders and further gene identification will help delineate these pathways.

Convergent evolution of adenosine aptamers spanning bacterial, human, and random sequences revealed by structure-based bioinformatics and genomic SELEX.

Aptamers are structured macromolecules in vitro evolved to bind molecular targets, whereas in nature they form the ligand-binding domains of riboswitches. Adenosine aptamers of a single structural family were isolated several times from random pools, but they have not been identified in genomic sequences. We used two unbiased methods, structure-based bioinformatics and human genome-based in vitro selection, to identify aptamers that form the same adenosine-binding structure in a bacterium, and several vertebrates, including humans. Two of the human aptamers map to introns of RAB3C and FGD3 genes. The RAB3C aptamer binds ATP with dissociation constants about 10 times lower than physiological ATP concentration, while the minimal FGD3 aptamer binds ATP only cotranscriptionally.

Crystal structure of inactive form of Rab3B.

Rab proteins are the largest family of ras-related GTPases in eukaryotic cells. They act as directional molecular switches at membrane trafficking, including vesicle budding, cargo sorting, transport, tethering, and fusion. Here, we generated and crystallized the Rab3B:GDP complex. The structure of the complex was solved to 1.9Å resolution and the structural base comparison with other Rab3 members provides a structural basis for the GDP/GTP switch in controlling the activity of small GTPase. The comparison of charge distribution among the members of Rab3 also indicates their different roles in vesicular trafficking.

Tctex-1, a novel interaction partner of Rab3D, is required for osteoclastic bone resorption.

Vesicular transport along microtubules must be strictly regulated to sustain the unique structural and functional polarization of bone-resorbing osteoclasts. However, the molecular mechanisms bridging these vesicle-microtubule interactions remain largely obscure. Rab3D, a member of the Rab3 subfamily (Rab3A/B/C/D) of small exocytotic GTPases, represents a core component of the osteoclastic vesicle transport machinery. Here, we identify a new Rab3D-interacting partner, Tctex-1, a light chain of the cytoplasmic dynein microtubule motor complex, by a yeast two-hybrid screen. We demonstrate that Tctex-1 binds specifically to Rab3D in a GTP-dependent manner and co-occupies Rab3D-bearing vesicles in bone-resorbing osteoclasts. Furthermore, we provide evidence that Tctex-1 and Rab3D intimately associate with the dynein motor complex and microtubules in osteoclasts. Finally, targeted disruption of Tctex-1 by RNA interference significantly impairs bone resorption capacity and mislocalizes Rab3D vesicles in osteoclasts, attesting to the notion that components of the Rab3D-trafficking pathway contribute to the maintenance of osteoclastic resorptive function.

Molecular anatomy of the hair cell's ribbon synapse.

Hearing depends on reliable and temporally precise neurotransmission by cochlear hair cells. The wide dynamic range and high sensitivity with which these cells encode acoustic stimuli are associated with a presynaptic specialization termed the presynaptic dense body or synaptic ribbon. Apposed to the presynaptic density, this spherical or flattened structure tethers a layer of synaptic vesicles and is thought to facilitate their exocytotic fusion. Although defining the molecular constituents of the hair cell's synaptic ribbon should contribute to our understanding of neurotransmitter release at this synapse, accomplishing this task has been slowed by the difficulty of obtaining sufficient amounts of starting material for protein analysis from hair cells. We isolated synaptic material from chicken cochleas, purified synaptic ribbons with specific immunological reagents, and identified the associated proteins by tandem mass spectrometry. Purification of the ribbons revealed a predominant composition of C-terminal-binding proteins, especially ribeye, in association with the small GTPase Rab3, which is possibly involved in attaching vesicles to the ribbon. In comparison with the components of conventional synapses and of retinal ribbon synapses, we observed that certain regulatory proteins are excluded from the hair cell's synapse. Using antisera against several of the novel proteins and membrane-trafficking components that we had identified, we documented their localization in isolated hair cells. Our results indicate that the ribbon synapses of hair cells display modifications to the presynaptic machinery that are associated with the high-fidelity transmission of acoustic signals to the brain.

Rab3-interacting molecule gamma isoforms lacking the Rab3-binding domain induce long lasting currents but block neurotransmitter vesicle anchoring in voltage-dependent P/Q-type Ca2+ channels.

Assembly of voltage-dependent Ca(2+) channels (VDCCs) with their associated proteins regulates the coupling of VDCCs with upstream and downstream cellular events. Among the four isoforms of the Rab3-interacting molecule (RIM1 to -4), we have previously reported that VDCC beta-subunits physically interact with the long alpha isoform of the presynaptic active zone scaffolding protein RIM1 (RIM1alpha) via its C terminus containing the C(2)B domain. This interaction cooperates with RIM1alpha-Rab3 interaction to support neurotransmitter exocytosis by anchoring vesicles in the vicinity of VDCCs and by maintaining depolarization-triggered Ca(2+) influx as a result of marked inhibition of voltage-dependent inactivation of VDCCs. However, physiological functions have not yet been elucidated for RIM3 and RIM4, which exist only as short gamma isoforms (gamma-RIMs), carrying the C-terminal C(2)B domain common to RIMs but not the Rab3-binding region and other structural motifs present in the alpha-RIMs, including RIM1alpha. Here, we demonstrate that gamma-RIMs also exert prominent suppression of VDCC inactivation via direct binding to beta-subunits. In the pheochromocytoma PC12 cells, this common functional feature allows native RIMs to enhance acetylcholine secretion, whereas gamma-RIMs are uniquely different from alpha-RIMs in blocking localization of neurotransmitter-containing vesicles near the plasma membrane. Gamma-RIMs as well as alpha-RIMs show wide distribution in central neurons, but knockdown of gamma-RIMs attenuated glutamate release to a lesser extent than that of alpha-RIMs in cultured cerebellar neurons. The results suggest that sustained Ca(2+) influx through suppression of VDCC inactivation by RIMs is a ubiquitous property of neurons, whereas the extent of vesicle anchoring to VDCCs at the plasma membrane may depend on the competition of alpha-RIMs with gamma-RIMs for VDCC beta-subunits.

ApRab3, a biosynthetic Rab protein, accumulates on the maturing phagosomes and symbiosomes in the tropical sea anemone, Aiptasia pulchella.

Symbiosome biogenesis and function are central to the endosymbiotic interaction between symbiotic dinoflagellates and their host cnidarians. To understand these important organelles, we have been conducting studies to identify and characterize symbiosome-associated proteins of the Rab family, key regulatory components of vesicular trafficking and membrane fusion in eukaryotic cells. Our prior studies have implicated three endocytic Rab proteins in the regulation of symbiosome biogenesis. Here, we show that ApRab3 is a new member of the Rab3 subfamily, associating with symbiosomes and accumulating on the maturing phagosomes in the A. pulchella digestive cells. ApRab3 is 78% identical to human Rab3C, and contains all Rab 3-specific signature motifs. EGFP-ApRab3-labeled vesicular structures tended to either align along the cell peripheral, or aggregate at one side of the nucleus. ApRab3 specifically co-distributed with the TGN marker, WGA, but not other organelle-specific markers tested. Immunofluorescence staining with a specific peptide antibody showed similar results. Significantly, an expression of a constitutively active mutant caused the enlargement and random dispersion of EGFP-ApRab3-decorated compartments in PC12 cells. Together, these data suggest that ApRab3 is a new member of the Rab3 subfamily, participating in the biosynthetic trafficking pathway, and symbiosome biogenesis involves an interaction with ApRab3-positive vesicles.

KIF1Bbeta- and KIF1A-mediated axonal transport of presynaptic regulator Rab3 occurs in a GTP-dependent manner through DENN/MADD.

Synaptic proteins are synthesized in the cell body and transported down the axon by microtubule-dependent motors. We previously reported that KIF1Bbeta and KIF1A motors are essential for transporting synaptic vesicle precursors; however the mechanisms that regulate transport, as well as cargo recognition and control of cargo loading and unloading remain largely unknown. Here, we show that DENN/MADD (Rab3-GEP) is an essential part of the regulation mechanism through direct interaction with the stalk domain of KIF1Bbeta and KIF1A. We also show that DENN/MADD binds preferentially to GTP-Rab3 and acts as a Rab3 effector. These molecular interactions are fundamental as sequential genetic perturbations revealed that KIF1Bbeta and KIF1A are essential for the transport of DENN/MADD and Rab3, whereas DENN/MADD is essential for the transport of Rab3. GTP-Rab3 was more effectively transported than GDP-Rab3, suggesting that the nucleotide state of Rab3 regulates axonal transport of Rab3-carrying vesicles through preferential interaction with DENN/MADD.

Interaction of Rab3B with microtubule-binding protein Gas8 in NIH 3T3 cells.

Rab3 subfamily small G proteins (Rab3A, Rab3B, Rab3C, and Rab3D) control the regulated exocytosis in neuronal/secretory cells. Rab3B is also detected and upregulated in non-neuronal/non-secretory cells, whereas its function remains elusive. In the present study, we identified growth-arrest-specific gene 8 (Gas8), an evolutionally conserved microtubule-binding protein that is upregulated in growth-arrested NIH 3T3 cells and involved in the dynein motor regulation in flagellar/ciliary axoneme, as a novel Rab3B-binding protein using a yeast two-hybrid system. Rab3B as well as Gas8 was upregulated in growth-arrested NIH 3T3 cells and enriched in testis and lung with well-developed flagella/cilia. Gas8 was specifically interacted with the GTP-bound form of Rab3B and co-localized with Rab3B at the Golgi in NIH 3T3 cells. Furthermore, Rab3B was relocated upon expression of the Rab3B-binding domain of Gas8. These results suggest that Gas8 links Rab3B to microtubules in NIH 3T3 cells.

Alternative splicing in the first alpha-helical region of the Rab-binding domain of Rim regulates Rab3A binding activity: is Rim a Rab3 effector protein during evolution?

Rim1 and Rim2 were originally described as specific Rab3A-effector proteins involved in the regulation of secretory vesicle exocytosis. The putative Rab3A-binding domain (RBD) of Rim consists of two alpha-helical regions (named RBD1 and RBD2) separated by two zinc finger motifs. Although alternative splicing in the RBD1 of Rim is known to produce long and short forms of RBD (named Rim1 and Rim1Delta56-105, and Rim2(+40A) and Rim2, respectively), with the long form of Rim1 and short form of Rim2 being dominant in mouse brain, the physiological significance of the alternative splicing in RBD1 has never been elucidated. In the present study I discovered that alternative splicing in Rim RBD1 alters Rab3A binding affinity to Rims, and found that insertion of 40 amino acids into the RBD1 of Rim2 (i.e. Rim2(+40A)) dramatically reduced its Rab3A binding activity (more than a 50-fold decrease in affinity). Similarly, Rim1Delta56-105 exhibited higher affinity binding to Rab3A than the long form of Rim1. Expression of the short forms of the Rim RBD in PC12 cells co-localized well with endogenous Rab3A, whereas expression of the long forms of the Rim RBD in PC12 cells resulted in cytoplasimc and nuclear localization. Moreover, I found that Caenorhabditis elegans Rim/UNC-10 (ce-Rim) and Drosophila Rim (dm-Rim) do not interact with ce-Rab3 and dm-Rab3, respectively, indicating that the Rab3-effector function of Rim has not been retained during evolution. Based on these findings, I propose that the Rab3A-effector function of Rim during secretory vesicle exocytosis is limited to the short form of the mammalian Rim RBD alone.

Modulation of secretory functions in epithelia by adenovirus capsid proteins.

To evaluate the safety of adenovirus-derived capsid proteins for ocular gene delivery, we have investigated their effects on the morphology and function of the acinar epithelial cells of the lacrimal gland. These cells are responsible for basal and stimulated release of proteins and electrolytes into ocular fluid, a process essential in maintaining the health of the ocular surface. Acinar epithelial cells from rabbit lacrimal gland were exposed to one of two adenovirus serotype 5 capsid proteins, penton or knob (the carboxy-terminal fragment of the fiber capsid protein). Sustained (16-18 h) exposure to the penton at 20 microg/ml was associated with major changes in the organization of the regulated secretory pathway and cytoskeleton. These changes included an apparent loss of mature secretory vesicles enriched in rab3D around the apical lumen as well as a depletion of apical actin. The microtubule array in penton-treated acini also exhibited bundling and disorganization. None of these effects were elicited by exposure to knob protein. Penton treatment also caused a significant (p < or = 0.05) increase and decrease in basal and carbachol-stimulated release, respectively, of bulk protein. Competition studies showed that RGD peptide partially prevented the penton-induced changes in rab3D-enriched secretory vesicles and actin filaments. These findings suggest that the adenovirus penton protein compromises normal acinar secretory compartment organization and function and that these changes are due at least partly to penton-integrin interactions.

A novel rabconnectin-3-binding protein that directly binds a GDP/GTP exchange protein for Rab3A small G protein implicated in Ca(2+)-dependent exocytosis of neurotransmitter.

BACKGROUND: Rab3A, a member of the Rab3 small G protein family, regulates Ca2+-dependent exocytosis of neurotransmitter. The cyclical activation and inactivation of Rab3A are essential for the Rab3A action in exocytosis. GDP-Rab3A is activated to GTP-Rab3A by Rab3 GDP/GTP exchange protein (Rab3 GEP) and GTP-Rab3A is inactivated to GDP-Rab3A by Rab3 GTPase-activating protein (Rab3 GAP). We have recently found a novel protein, named rabconnectin-3, which is co-immunoprecipitated with Rab3 GEP or GAP from the extract of the crude synaptic vesicle (CSV) fraction of rat brain. Rabconnectin-3 is abundantly expressed in the brain where it is associated with synaptic vesicles. We have found that two more proteins are co-immunoprecipitated with Rab3 GEP from the CSV fraction of rat brain. We attempted here to isolate and characterize one of them. RESULTS: We determined its partial amino acid sequence, cloned its cDNA from a human cDNA library, and determined its primary structure. The protein consisted of 1490 amino acids (aa) and showed a calculated molecular weight of 163808. The protein had 7 WD domains. The protein was abundantly expressed in the brain where it co-localized with rabconnectin-3 on synaptic vesicles. The protein formed a stable complex with rabconnectin-3. We named this protein rabconnectin-3beta and renamed rabconnectin-3 rabconnectin-3alpha. Rabconnectin-3beta, but not rabconnectin-3alpha, directly bound Rab3 GEP. Neither rabconnectin-3alpha nor -3beta directly bound Rab3 GAP. CONCLUSION: These results indicate that rabconnectin-3 consists of the alpha and beta subunits and binds directly Rab3 GEP through the beta subunit and indirectly Rab3 GAP through an unidentified molecule(s).

Distinct Rab binding specificity of Rim1, Rim2, rabphilin, and Noc2. Identification of a critical determinant of Rab3A/Rab27A recognition by Rim2.

Rabphilin, Rim, and Noc2 have generally been believed to be the Rab3 isoform (Rab3A/B/C/D)-specific effectors that regulate secretory vesicle exocytosis in neurons and in some endocrine cells. The results of recent genetic analysis of rabphilin knock-out animals, however, strongly refute this notion, because there are no obvious genetic interactions between Rab3 and rabphilin in nematoda (Staunton, J., Ganetzky, B., and Nonet, M. L. (2001) J. Neurosci. 21, 9255-9264), suggesting that Rab3 is not a major ligand of rabphilin in vivo. In this study, I tested the interaction of rabphilin, Rim1, Rim2, and Noc2 with 42 different Rab proteins by cotransfection assay and found differences in rabphilin, Rim1, Rim2, and Noc2 binding to several Rab proteins that belong to the Rab functional group III (Rab3A/B/C/D, Rab26, Rab27A/B, and Rab37) and/or VIII (Rab8A and Rab10). Rim1 interacts with Rab3A/B/C/D, Rab10, Rab26, and Rab37; Rim2 interacts with Rab3A/B/C/D and Rab8A; and rabphilin and Noc2 interact with Rab3A/B/C/D, Rab8A, and Rab27A/B. By contrast, the synaptotagmin-like protein homology domain of Slp homologue lacking C2 domains-a (Slac2-a)/melanophilin specifically recognizes Rab27A/B but not other Rabs. I also found that alternative splicing events in the first alpha-helical region (alpha(1)) of the Rab binding domain of Rim1 alter the Rab binding specificity of Rim1. Site-directed mutagenesis and chimeric analyses of Rim2 and Slac2-a indicate that the acidic cluster (Glu-50, Glu-51, and Glu-52) in the alpha(1) region of the Rab binding domain of Rim2, which is not conserved in the synaptotagmin-like pro tein homology domain of Slac2-a, is a critical determinant of Rab3A recognition. Based on these results, I propose that Rim, rabphilin, and Noc2 function differently in concert with functional group III and/or VIII Rab proteins, including Rab3 isoforms.

Synaptotagmin-like protein (Slp) homology domain 1 of Slac2-a/melanophilin is a critical determinant of GTP-dependent specific binding to Rab27A.

The N-terminal synaptotagmin-like protein (Slp) homology domain (SHD) of the Slp and Slac2 families has recently been identified as a specific Rab27A-binding domain (Kuroda, T. S., Fukuda, M., Ariga, H., and Mikoshiba, K. (2002) J. Biol. Chem. 277, 9212-9218; Fukuda, M., Kuroda, T. S., and Mikoshiba, K. (2002) J. Biol. Chem. 277, 12432-12436). The SHD consists of two conserved alpha-helical regions (SHD1 and SHD2) that are often separated by two zinc finger motifs. However, the structural basis of Rab27A recognition by the SHD (i.e. involvement of each region (SHD1, zinc finger motifs, and SHD2) in Rab27A recognition and critical residue(s) for Rab27A/SHD interaction) had never been elucidated. In this study, systematic deletion analysis and Ala-based site-directed mutagenesis showed that SHD1 of Slac2-a/melanophilin alone is both necessary and sufficient for high affinity specific recognition of the GTP-bound form of Rab27A. By contrast, the zinc finger motifs and SHD2 are not an autonomous Rab27A-binding site and seem to be important for stabilization of the structure of the SHD or higher affinity Rab27A binding. In addition, chimeric analysis of Rab3A and Rab27A showed that the specific sequence of the switch II region of Rab27 isoforms (especially Leu-84, Phe-88, and Asp-91 of Rab27A), which is not conserved in the Rab3 or Rab8 isoforms, is essential for recognition by the Slac2-a SHD. Based on these findings, I propose that SHD1 of the Slp and Slac2 families be referred to as RBD27 (Rab-binding domain specific for Rab27 isoforms).

The death domain of Rab3 guanine nucleotide exchange protein in GDP/GTP exchange activity in living cells.

Rab3 GTPases regulate exocytosis of neurons, endocrine and exocrine cells. In the present paper, we report a system to measure the guanine nucleotide status of Rab3 proteins in living cells. The assay is based on the ability of the Rab3 interacting molecule RIM to extract selectively the GTP-bound form of Rab3. Using this system, we found that approx. 20% of wild-type Rab3A, -B, -C or -D transfected in the insulin-secreting cell line HIT-T15 is in the GTP-bound conformation. The pool of activated Rab3 is decreased under conditions that stimulate exocytosis or by co-expression of the Rab3 GTPase-activating protein. In contrast, co-expression of Mss4 or Rab3-GEP (guanine nucleotide exchange protein) increases by approx. 3-fold the GTP-bound pool of Rab3 isoforms. Rab3-GEP is very similar to MADD, a death domain-containing protein that associates with the type 1 tumour necrosis factor receptor. We observed that the death domain of Rab3-GEP is involved in intramolecular interactions and that deletions or mutations that affect this domain of the protein impair the nucleotide exchange activity towards Rab3. We propose that the death domain of Rab3-GEP acts as a molecular switch and co-ordinates multiple functions of the protein by exchanging its binding partners.