Cell biology. Fusion without SNAREs?

Highly orchestrated molecular rearrangements are required for two membranes to fuse, as happens, for example, during neurotransmitter release into the synapse. In an elegant Perspective, Scales et al. discuss two studies (Schoch et al., Wang et al.) that shed new light on the protein interactions involved in membrane fusion.

The Di-leucine motif of vesicle-associated membrane protein 4 is required for its localization and AP-1 binding.

Heterotetrameric adaptor complexes and SNAREs play key roles in the specificity of membrane budding and fusion. Here we test the hypothesis that vesicle budding and membrane fusion are coupled by the interaction of these molecules. We investigate the role of the di-leucine motif of vesicle-associated membrane protein 4 (VAMP4) in adaptor binding and localization of VAMP4. Mutation of the di-leucine motif inhibits AP-1 binding in vitro and affects the steady state distribution of VAMP4 in vivo.

Crystal structure and biophysical properties of a complex between the N-terminal SNARE region of SNAP25 and syntaxin 1a.

SNARE proteins are required for intracellular membrane fusion. In the neuron, the plasma membrane SNAREs syntaxin 1a and SNAP25 bind to VAMP2 found on neurotransmitter-containing vesicles. These three proteins contain "SNARE regions" that mediate their association into stable tetrameric coiled-coil structures. Syntaxin 1a contributes one such region, designated H3, and SNAP25 contributes two SNARE regions to the fusogenic complex with VAMP2. Syntaxin 1a H3 (syn1aH3) and SNAP25 can form a stable assembly, which can then be bound by VAMP2 to form the full SNARE complex. Here we show that syn1aH3 can also form a stable but kinetically trapped complex with the N-terminal SNARE region of SNAP25 (S25N). The crystal structure of this complex reveals an extended parallel four-helix bundle similar to that of the core SNARE and the syn1aH3-SNAP25 complexes. The inherent ability of syn1aH3 and S25N to associate stably in vitro implies that the intracellular fusion machinery must prevent formation of, or remove, any non-productive complexes. Comparison with the syn1aH3-SNAP25 complex suggests that the linkage of the N- and C-terminal SNAP25 SNARE regions is kinetically advantageous in preventing formation of the non-productive syn1aH3-S25N complex. We also demonstrate that the syn1aH3-S25N complex can be disassembled by alpha-SNAP and N-ethylmaleimide-sensitive factor.

The Sec6/8 complex in mammalian cells: characterization of mammalian Sec3, subunit interactions, and expression of subunits in polarized cells.

The yeast exocyst complex (also called Sec6/8 complex in higher eukaryotes) is a multiprotein complex essential for targeting exocytic vesicles to specific docking sites on the plasma membrane. It is composed of eight proteins (Sec3, -5, -6, -8, -10, and -15, and Exo70 and -84), with molecular weights ranging from 70 to 144 kDa. Mammalian orthologues for seven of these proteins have been described and here we report the cloning and initial characterization of the remaining subunit, Sec3. Human Sec3 (hSec3) shares 17% sequence identity with yeast Sec3p, interacts in the two-hybrid system with other subunits of the complex (Sec5 and Sec8), and is expressed in almost all tissues tested. In yeast, Sec3p has been proposed to be a spatial landmark for polarized secretion (1), and its localization depends on its interaction with Rho1p (2). We demonstrate here that hSec3 lacks the potential Rho1-binding site and GFP-fusions of hSec3 are cytosolic. Green fluorescent protein (GFP)-fusions of nearly every subunit of the mammalian Sec6/8 complex were expressed in Madin-Darby canine kidney (MDCK) cells, but they failed to assemble into a complex with endogenous proteins and localized in the cytosol. Of the subunits tested, only GFP-Exo70 localized to lateral membrane sites of cell-cell contact when expressed in MDCK cells. Cells overexpressing GFP-Exo70 fail to form a tight monolayer, suggesting the Exo70 targeting interaction is critical for normal development of polarized epithelial cells.

Identification of a novel Rab11/25 binding domain present in Eferin and Rip proteins.

Rab11, a low molecular weight GTP-binding protein, has been shown to play a key role in a variety of cellular processes, including endosomal recycling, phagocytosis, and transport of secretory proteins from the trans-Golgi network. In this study we have described a novel Rab11 effector, EF-hands-containing Rab11-interacting protein (Eferin). In addition, we have identified a 20-amino acid domain that is present at the C terminus of Eferin and other Rab11/25-interacting proteins, such as Rip11 and nRip11. Using biochemical techniques we have demonstrated that this domain is necessary and sufficient for Rab11 binding in vitro and that it is required for localization of Rab11 effector proteins in vivo. The data suggest that various Rab effectors compete with each other for binding to Rab11/25 possibly accounting for the diversity of Rab11 functions.

Developmental regulation and specific brain distribution of phosphorabphilin.

Protein kinases and phosphatases play an important role in modulating synaptic transmission. The synaptic protein rabphilin associates with synaptic vesicles through the small GTPase Rab3A, binds Ca(2+) and phospholipids, and interacts with cytoskeletal elements, yet its function remains controversial. In this study, we have generated phosphospecific antibodies and studied the developmental, subcellular, and brain distribution of rabphilin phosphorylated at serine-234 and serine-274. Our results show that phosphorabphilin is present in vivo under basal conditions in a specific subset of synapses. The phosphorylated rabphilin is abundant in the cerebellum, midbrain, and medulla; phosphorabphilin is specifically enriched in the climbing fiber synapses of the cerebellar cortex. Its developmental profile reveals a sharp and transient increase at approximately postnatal day 16, a period critical for the activity-dependent pruning of supernumerary climbing fibers in the cerebellum. We propose that the phosphorylation of rabphilin regulates neuronal activity through development and in a synapse-specific manner.

Physiological modulation of rabphilin phosphorylation.

The dynamic modulation of protein function by phosphorylation plays an important role in regulating synaptic plasticity. Several proteins involved in synaptic transmission have been shown to be targets of protein kinases and phosphatases. A thorough analysis of the physiological role of these modifications has been hampered by the lack of reagents that specifically recognize the phosphorylated states of these proteins. In this study we analyze the physiological modulation of rabphilin using phosphospecific antibodies. We show that phosphorylation on serine-234 and serine-274 of rabphilin is dynamically regulated both under basal and stimulated conditions by the activity of kinases and phosphatases. The two sites are differentially phosphorylated by the stimulation of various kinases, suggesting a possible convergence of different pathways to modulate the function of the protein. Maximal stimulation was observed under plasma membrane-depolarizing conditions that trigger synaptic vesicle exocytosis. The increase in phosphorylation was critically dependent on external Ca(2+) and on the presence of Rab3a, a small GTPase that recruits rabphilin to synaptic vesicles. The rapid phosphorylation and dephosphorylation during and after stimulation demonstrates the transient nature of the modification. Our results indicate that rabphilin is phosphorylated on synaptic vesicles by Ca(2+)-dependent kinases that become active in synaptic terminals during exocytosis. We have found that phosphorabphilin has a reduced affinity for membranes; we therefore propose that the modulation of the membrane association of rabphilin has a role in the synaptic vesicle life cycle, perhaps in vesicle mobilization in preparation for subsequent rounds of neurotransmission.

Three SNARE complexes cooperate to mediate membrane fusion.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins of the syntaxin, SNAP-25, and VAMP families mediate intracellular membrane fusion through the formation of helical bundles that span opposing membranes. Soluble SNARE domains that lack their integral membrane anchors inhibit membrane fusion by forming nonfunctional complexes with endogenous SNARE proteins. In this study we investigate the dependence of membrane fusion on the concentration of a soluble SNARE coil domain derived from VAMP2. The increase in the inhibition of fusion observed with increasing concentration of inhibitor is best fit to a function that suggests three SNARE complexes cooperate to mediate fusion of a single vesicle. These three complexes likely contribute part of a protein and lipidic fusion pore.

Sequential SNARE assembly underlies priming and triggering of exocytosis.

Changes in SNARE conformations during MgATP-dependent priming of cracked PC12 cells were probed by their altered accessibility to various inhibitors. Dominant negative soluble syntaxin and, to a much lesser extent, VAMP coil domains inhibited exocytosis more efficiently after priming. Neurotoxins and an anti-SNAP25 antibody inhibited exocytosis less effectively after priming. We propose that SNAREs partially and reversibly assemble during priming, and that the syntaxin H3 domain is prevented from fully joining the complex until the arrival of the Ca2+ trigger. Furthermore, we find that mutation of hydrophobic residues of the SNAP25 C-terminal coil that contribute to SNARE core interactions affects the maximal rate of exocytosis, while mutation of charged residues on the surface of the complex affects the apparent affinity of the coil domain for the partially assembled complex.

Calcium regulation of exocytosis in PC12 cells.

The calcium (Ca(2+)) regulation of neurotransmitter release is poorly understood. Here we investigated several aspects of this process in PC12 cells. We first showed that osmotic shock by 1 m sucrose stimulated rapid release of neurotransmitters from intact PC12 cells, indicating that most of the vesicles were docked at the plasma membrane. Second, we further investigated the mechanism of rescue of botulinum neurotoxin E inhibition of release by recombinant SNAP-25 COOH-terminal coil, which is known to be required in the triggering stage. We confirmed here that Ca(2+) was required simultaneously with the SNAP-25 peptide, with no significant increase in release if either the peptide or Ca(2+) was present during the priming stage as well as the triggering, suggesting that SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) complex assembly was involved in the final Ca(2+)-triggered event. Using this rescue system, we also identified a series of acidic surface SNAP-25 residues that rescued better than wild-type when mutated, due to broadened Ca(2+) sensitivity, suggesting that this charged patch may interact electrostatically with a negative regulator of membrane fusion. Finally, we showed that the previously demonstrated stimulation of exocytosis in this system by calmodulin required calcium binding, since calmodulin mutants defective in Ca(2+)-binding were not able to enhance release.

A discontinuous SNAP-25 C-terminal coil supports exocytosis.

Membrane fusion requires the formation of four-helical bundles comprised of the SNARE proteins syntaxin, vesicle-associated membrane protein (VAMP), and the synaptosomal-associated protein of 25 kDa (SNAP-25). Botulinum neurotoxin E cleaves the C-terminal coil of SNAP-25, inhibiting exocytosis of norepinephrine from permeabilized PC12 cells. Addition of a 26-mer peptide comprising the C terminus of SNAP-25 that is cleaved by the toxin restores exocytosis, demonstrating that continuity of the SNAP-25 C-terminal helix is not critical for its function. By contrast, vesicle-associated membrane protein peptides could not rescue botulinum neurotoxin D-treated cells, suggesting that helix continuity is critical for VAMP function. Much higher concentrations of the SNAP-25 C-terminal peptide are required for rescuing exocytosis (K(assembly) = approximately 460 microm) than for binding to other SNAREs in vitro (Kd < 5 microm). Each residue of the peptide was mutated to alanine to assess its functional importance. Whereas most mutants rescue exocytosis with lower efficiency than the wild type peptide, D186A rescues with higher efficiency, and kinetic analysis suggests this is because of higher affinity for the cellular binding site. This is consistent with Asp-186 contributing to negative regulation of the fusion process.

A novel snare N-terminal domain revealed by the crystal structure of Sec22b.

Intra-cellular membrane fusion is facilitated by the association of SNAREs from opposite membranes into stable alpha-helical bundles. Many SNAREs, in addition to their alpha-helical regions, contain N-terminal domains that likely have essential regulatory functions. To better understand this regulation, we have determined the 2.4-A crystal structure of the 130-amino acid N-terminal domain of mouse Sec22b (mSec22b), a SNARE involved in endoplasmic reticulum/Golgi membrane trafficking. The domain consists of a mixed alpha-helical/beta-sheet fold that resembles a circular permutation of the actin/poly-proline binding protein, profilin, and the GAF/PAS family of regulatory modules. The structure is distinct from the previously characterized N-terminal domain of syntaxin 1A, and, unlike syntaxin 1A, the N-terminal domain of mSec22b has no effect on the rate of SNARE assembly in vitro. An analysis of surface conserved residues reveals a potential protein interaction site. Key residues in this site are distinct in two mammalian Sec22 variants that lack SNARE domains. Finally, sequence analysis indicates that a similar domain is likely present in the endosomal/lysosomal SNARE VAMP7.

SNARE-mediated membrane fusion.

SNARE proteins have been proposed to mediate all intracellular membrane fusion events. There are over 30 SNARE family members in mammalian cells and each is found in a distinct subcellular compartment. It is likely that SNAREs encode aspects of membrane transport specificity but the mechanism by which this specificity is achieved remains controversial. Functional studies have provided exciting insights into how SNARE proteins interact with each other to generate the driving force needed to fuse lipid bilayers.

A genomic perspective on membrane compartment organization.

Now that whole genome sequences are available for many eukaryotic organisms from yeast to man, we can form broad hypotheses on the basis of the relative expansion of protein families. To investigate the molecular mechanisms responsible for the organization of membrane compartments, we identified members of the SNARE, coat complex, Rab and Sec1 protein families in four eukaryotic genomes. Of these families only the Rab family expanded from the unicellular yeast to the multicellular fly and worm. All families were expanded in humans, where we find 35 SNAREs, 60 Rabs and 53 coat complex subunits. In addition, we were able to resolve the SNARE class of proteins into four distinct subfamilies.

Self-association of the H3 region of syntaxin 1A. Implications for intermediates in SNARE complex assembly.

Intracellular membrane fusion requires SNARE proteins found on the vesicle and target membranes. SNAREs associate by formation of a parallel four-helix bundle, and it has been suggested that formation of this complex promotes membrane fusion. The membrane proximal region of the cytoplasmic domain of the SNARE syntaxin 1A, designated H3, contributes one of the four helices to the SNARE complex. In the crystal structure of syntaxin 1A H3, four molecules associate as a homotetramer composed of two pairs of parallel helices that are anti-parallel to each other. The H3 oligomer observed in the crystals is also found in solution, as assessed by gel filtration and chemical cross-linking studies. The crystal structure reveals that the highly conserved Phe-216 packs against conserved Gln-226 residues present on the anti-parallel pair of helices. Modeling indicates that Phe-216 prevents parallel tetramer formation. Mutation of Phe-216 to Leu appears to allow formation of parallel tetramers, whereas mutation to Ala destabilizes the protein. These results indicate that Phe-216 has a role in preventing formation of stable parallel helical bundles, thus favoring the interaction of the H3 region of syntaxin 1a with other proteins involved in membrane fusion.

The ionic layer is required for efficient dissociation of the SNARE complex by alpha-SNAP and NSF.

The four-helical bundle soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor (SNARE) complex that mediates intracellular membrane fusion events contains a highly conserved ionic layer at the center of an otherwise hydrophobic core. This layer has an undetermined function; it consists of glutamine (Q) residues in syntaxin and the two synaptosomal-associated protein of 25 kDa (SNAP-25) family helices, and an arginine (R) in vesicle-associated membrane protein (a 3Q:1R ratio). Here, we show that the ionic-layer glutamine of syntaxin is required for efficient alpha-SNAP and NSF-mediated dissociation of the complex. When this residue is mutated, the SNARE complex still binds to alpha-SNAP and NSF and is released through ATP hydrolysis by NSF, but the complex no longer dissociates into SNARE monomers. Thus, one function of the ionic layer--in particular, the glutamine of syntaxin--is to couple ATP hydrolysis by NSF to the dissociation of the fusion complex. We propose that alpha-SNAP and NSF drive conformational changes at the ionic layer through specific interactions with the syntaxin glutamine, resulting in the dissociation of the SNARE complex.

Molecular determinants of the functional interaction between syntaxin and N-type Ca2+ channel gating.

Syntaxin is a key presynaptic protein that binds to N- and P/Q-type Ca(2+) channels in biochemical studies and affects gating of these Ca(2+) channels in expression systems and in synaptosomes. The present study was aimed at understanding the molecular basis of syntaxin modulation of N-type channel gating. Mutagenesis of either syntaxin 1A or the pore-forming alpha(1B) subunit of N-type Ca(2+) channels was combined with functional assays of N-type channel gating in a Xenopus oocyte coexpression system and in biochemical binding experiments in vitro. Our analysis showed that the transmembrane region of syntaxin and a short region within the H3 helical cytoplasmic domain of syntaxin, containing residues Ala-240 and Val-244, appeared critical for the channel modulation but not for biochemical association with the "synprint site" in the II/III loop of alpha(1B). These results suggest that syntaxin and the alpha(1B) subunit engage in two kinds of interactions: an anchoring interaction via the II/III loop synprint site and a modulatory interaction via another site located elsewhere in the channel sequence. The segment of syntaxin H3 found to be involved in the modulatory interaction would lie hidden within the four-helix structure of the SNARE complex, supporting the hypothesis that syntaxin's ability to regulate N-type Ca(2+) channels would be enabled after SNARE complex disassembly after synaptic vesicle exocytosis.

Mechanisms of synaptic vesicle exocytosis.

Chemical synaptic transmission serves as the main form of cell to cell communication in the nervous system. Neurotransmitter release occurs through the process of regulated exocytosis, in which a synaptic vesicle releases its contents in response to an increase in calcium. The use of genetic, biochemical, structural, and functional studies has led to the identification of factors important in the synaptic vesicle life cycle. Here we focus on the prominent role of SNARE (soluble NSF attachment protein receptor) proteins during membrane fusion and the regulation of SNARE function by Rab3a, nSec1, and NSF. Many of the proteins important for transmitter release have homologs involved in intracellular vesicle transport, and all forms of vesicle trafficking share common basic principles. Finally, modifications to the synaptic exocytosis pathway are very likely to underlie certain forms of synaptic plasticity and therefore contribute to learning and memory.

Syntaxin 17 is abundant in steroidogenic cells and implicated in smooth endoplasmic reticulum membrane dynamics.

The endoplasmic reticulum (ER) consists of subcompartments that have distinct protein constituents, morphological appearances, and functions. To understand the mechanisms that regulate the intricate and dynamic organization of the endoplasmic reticulum, it is important to identify and characterize the molecular machinery involved in the assembly and maintenance of the different subcompartments. Here we report that syntaxin 17 is abundantly expressed in steroidogenic cell types and specifically localizes to smooth membranes of the ER. By immunoprecipitation analyses, syntaxin 17 exists in complexes with a syntaxin regulatory protein, rsly1, and/or two intermediate compartment SNARE proteins, rsec22b and rbet1. Furthermore, we found that syntaxin 17 is anchored to the smooth endoplasmic reticulum through an unusual mechanism, requiring two adjacent hydrophobic domains near its carboxyl terminus. Converging lines of evidence indicate that syntaxin 17 functions in a vesicle-trafficking step to the smooth-surfaced tubular ER membranes that are abundant in steroidogenic cells.