SNAP-29 is a promiscuous syntaxin-binding SNARE.

SNARE proteins are key regulators of membrane fusion and are proposed to dictate the specificity with which particular vesicles fuse with particular target organelles. On intracellular organelles that serve as targets for transport vesicles, organelle-specific syntaxins form heterodimers with either SNAP-23 or its recently described homolog SNAP-29. We have performed a variety of in vitro and in vivo binding assays in an attempt to determine whether SNAP-23 and SNAP-29 differ in their ability to form binary SNARE complexes with different intracellular syntaxins. While SNAP-23 preferentially binds to plasma membrane-localized syntaxins, SNAP-29 binds to both plasma membrane and intracellular syntaxins equally well. Furthermore, binding to SNAP-29 augments the ability of syntaxin to bind to vesicle-associated SNAREs and the presence of vesicle SNAREs dramatically increases SNAP-29 binding to syntaxin. These data suggest that SNAP-23 preferentially regulates plasma membrane-vesicle fusion events while SNAP-29 plays a role in the maintenance of various intracellular protein trafficking pathways.     

An activated Q‐SNARE/SM protein complex as a possible intermediate in SNARE assembly

Assembly of the SNARE proteins syntaxin1, SNAP25, and synaptobrevin into a SNARE complex is essential for exocytosis in neurons. For efficient assembly, SNAREs interact with additional proteins but neither the nature of the intermediates nor the sequence of protein assembly is known. Here, we have characterized a ternary complex between syntaxin1, SNAP25, and the SM protein Munc18‐1 as a possible acceptor complex for the R‐SNARE synaptobrevin. The ternary complex binds synaptobrevin with fast kinetics, resulting in the rapid formation of a fully zippered SNARE complex to which Munc18‐1 remains tethered by the N‐terminal domain of syntaxin1. Intriguingly, only one of the synaptobrevin truncation mutants (Syb1‐65) was able to bind to the syntaxin1:SNAP25:Munc18‐1 complex, suggesting either a cooperative zippering mechanism that proceeds bidirectionally or the progressive R‐SNARE binding via an SM template. Moreover, the complex is resistant to disassembly by NSF. Based on these findings, we consider the ternary complex as a strong candidate for a physiological intermediate in SNARE assembly.     

Folding intermediates of SNARE complex assembly

SNARE (soluble NSF attachment protein receptor) proteins assemble into a stable complex essential for vesicle-membrane fusion. To further understand SNARE function we have used solution nuclear magnetic resonance (NMR) spectroscopy to characterize three assembly states of a yeast SNARE complex: first, the 'closed' conformation of Sso1; second, the binary complex of Sso1 and Sec9; and third, the ternary complex of Sso1, Sec9 and Snc1. Sec9 and Snc1 are unstructured in isolation. Sso1 likely consists of a four helix bundle formed by part of the C-terminal Hcore domain and the N-terminal H(A)H(B)H(C) domain, and this bundle is flanked on both sides by large flexible regions. Sso1 switches to an 'open' state when its Hcore domain binds Sec9. Conformational switching of the Hcore domain, via H(A)H(B)H(C), may provide a key regulatory mechanism in SNARE assembly. Formation of binary and ternary complexes induces additional alpha-helical structure in previously unstructured regions. Our data suggest a directed assembly process beginning distal to the membrane surfaces and proceeding toward them, bringing membranes into close proximity and possibly leading to membrane fusion.     

Immunohistochemical localization in human tissues of GPI-80, a novel glycosylphosphatidyl inositol-anchored protein that may regulate neutrophil extravasation

We molecular-cloned a novel 80-kDa human glycosyl-phosphatidyl inositol (GPI)-anchored protein, designated GPI-80, that may regulate neutrophil extravasation. To identify the possible role of GPI-80 in vivo, we examined the immunohistochemical localization of GPI-80 in various human tissues. Our data show that GPI-80 is mainly located in polymorphonuclear leukocytes (PMNs), endothelial cells of the vessels, parietal cells and mucous neck cells of the stomach, goblet cells of the jejunum, and alveolar macrophages of the lung. The pathomechanisms of these positive findings in the gastric glands and the intestinal glands are not well elucidated and further studies will be needed.     

Interactions within the yeast t-SNARE Sso1p that control SNARE complex assembly

In the eukaryotic secretory and endocytic pathways, transport vesicles shuttle cargo among intracellular organelles and to and from the plasma membrane. Cargo delivery entails fusion of the transport vesicle with its target, a process thought to be mediated by membrane bridging SNARE protein complexes. Temporal and spatial control of intracellular trafficking depends in part on regulating the assembly of these complexes. In vitro, SNARE assembly is inhibited by the closed conformation adopted by the syntaxin family of SNAREs. To visualize this closed conformation directly, the X-ray crystal structure of a yeast syntaxin, Sso1p, has been determined and refined to 2.1 A resolution. Mutants designed to destabilize the closed conformation exhibit accelerated rates of SNARE assembly. Our results provide insight into the mechanism of SNARE assembly and its intramolecular and intermolecular regulation.     

Syntaphilin: a syntaxin-1 clamp that controls SNARE assembly

Syntaxin-1 is a key component of the synaptic vesicle docking/fusion machinery that forms the SNARE complex with VAMP/synaptobrevin and SNAP-25. Identifying proteins that modulate SNARE complex formation is critical for understanding the molecular mechanisms underlying neurotransmitter release and its modulation. We have cloned and characterized a protein called syntaphilin that is selectively expressed in brain. Syntaphilin competes with SNAP-25 for binding to syntaxin-1 and inhibits SNARE complex formation by absorbing free syntaxin-1. Transient overexpression of syntaphilin in cultured hippocampal neurons significantly reduces neurotransmitter release. Furthermore, introduction of syntaphilin into presynaptic superior cervical ganglion neurons in culture inhibits synaptic transmission. These findings suggest that syntaphilin may function as a molecular clamp that controls free syntaxin-1 availability for the assembly of the SNARE complex, and thereby regulates synaptic vesicle exocytosis.     

The N-terminal domains of syntaxin 7 and vti1b form three-helix bundles that differ in their ability to regulate SNARE complex assembly

The SNAREs syntaxin 7, syntaxin 8, vti1b, and endobrevin/VAMP8 function in the fusion of late endosomes. Although the core complex formed by these SNAREs is very similar to the neuronal SNARE complex, it differs from the neuronal complex in that three of the four SNAREs contain extended N-terminal regions of unknown structure and function. Here we show that the N-terminal regions of syntaxin 7, syntaxin 8, and vti1b contain well folded alpha-helical domains. Multidimensional NMR spectroscopy revealed that in syntaxin 7 and vti1b, the domains form three-helix bundles resembling those of syntaxin 1, Sso1p, and Vam3p. The three-helix bundle domain of vti1b is the first of its kind identified in a SNARE outside the syntaxin family. Only syntaxin 7 adopts a closed conformation, whereas in vti1b and syntaxin 8, the N-terminal domains do not interact with the adjacent SNARE motifs. Accordingly, the rate of SNARE complex assembly is retarded about 7-fold when syntaxin 7 contains its N-terminal domain, whereas the N-terminal domains of vti1b and syntaxin 8 have no influence on assembly kinetics. We conclude that three-helix bundles represent a common fold for SNARE N-terminal domains, not restricted to the syntaxin family. However, they differ in their ability to adopt closed conformations and thus to regulate the assembly of SNARE complexes.     

Sec17p and HOPS, in distinct SNARE complexes, mediate SNARE complex disruption or assembly for fusion

SNARE functions during membrane docking and fusion are regulated by Sec1/Munc18 (SM) chaperones and Rab/Ypt GTPase effectors. These functions for yeast vacuole fusion are combined in the six-subunit HOPS complex. HOPS facilitates Ypt7p nucleotide exchange, is a Ypt7p effector, and contains an SM protein. We have dissected the associations and requirements for HOPS, Ypt7p, and Sec17/18p during SNARE complex assembly. Vacuole SNARE complexes bind either Sec17p or the HOPS complex, but not both. Sec17p and its co-chaperone Sec18p disassemble SNARE complexes. Ypt7p regulates the reassembly of unpaired SNAREs with each other and with HOPS, forming HOPS.SNARE complexes prior to fusion. After HOPS.SNARE assembly, lipid rearrangements are still required for vacuole content mixing. Thus, Sec17p and HOPS have mutually exclusive interactions with vacuole SNAREs to mediate disruption of SNARE complexes or their assembly for docking and fusion. Sec17p may displace HOPS from SNAREs to permit subsequent rounds of fusion.     

SNARE assembly and membrane fusion, a kinetic analysis

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) assembly may promote intracellular membrane fusion, an essential process for vesicular transport in cells. Core complex formation between vesicle-associated SNARE and target membrane SNARE perhaps drives the merging of two membranes into a single bilayer. Using spin-labeling EPR, trans-SNARE complex formation was monitored "locally" at four different core locations of recombinant yeast SNAREs, which are individually reconstituted into phospholipid vesicles. The results indicate that the time scales of core formation are virtually the same at all four locations throughout the core region, indicating the possibility of a single step core assembly, which appears to be somewhat different from what has been postulated by the "zipper" model. The EPR data were then compared with the kinetics of the lipid mixing measured with the fluorescence assay. The analysis suggests that SNARE core assembly occurs on a much faster time scale than the lipid mixing, providing a new insight into the timing of individual events in SNARE-induced membrane fusion.     

SNARE complex assembly is required for human sperm acrosome reaction

Exocytosis of the acrosome (the acrosome reaction) is a terminal morphological alteration that sperm must undergo prior to penetration of the extracellular coat of the egg. Ca(2+) is an essential mediator of this regulated secretory event. Aided by a streptolysin-O permeabilization protocol developed in our laboratory, we have previously demonstrated requirements for Rab3A, NSF, and synaptotagmin VI in the human sperm acrosome reaction. Interestingly, Rab3A elicits an exocytotic response of comparable magnitude to that of Ca(2+). Here, we report a direct role for the SNARE complex in the acrosome reaction. First, the presence of SNARE proteins is demonstrated by Western blot. Second, the Ca(2+)-triggered acrosome reaction is inhibited by botulinum neurotoxins BoNT/A, -E, -C, and -F. Third, antibody inhibition studies show a requirement for SNAP-25, SNAP-23, syntaxins 1A, 1B, 4, and 6, and VAMP 2. Fourth, addition of bacterially expressed SNAP-25 and SNAP-23 abolishes exocytosis. Acrosome reaction elicited by Rab3-GTP is also inhibited by BoNT/A, -C, and -F. Taken together, these results demonstrate a requirement for members of all SNARE protein families in the Ca(2+)- and Rab3A-triggered acrosome reaction. Furthermore, they indicate that the onset of sperm exocytosis relies on the functional assembly of SNARE complexes.     

A spinal muscular atrophy modifier implicates the SMN protein in SNARE complex assembly at neuromuscular synapses

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Mdp3 is a novel microtubule-binding protein that regulates microtubule assembly and stability

Microtubule-binding proteins are a group of molecules that associate with microtubules, regulate the structural properties of microtubules, and thereby participate in diverse microtubule-mediated cellular activities. A recent mass spectrometry-based proteomic study has identified microtubule-associated protein 7 (MAP7) domain-containing 3 (Mdp3) as a potential microtubule-binding protein. However, its subcellular localization and functional importance are not characterized. In this study, by GST-pulldown assays, we found that Mdp3 interacted with tubulin both in cells and in vitro. Immunofluorescence microscopy and microtubule cosedimentation assays revealed that Mdp3 also associated with microtubules. Serial deletion experiments showed that the two coiled coil motifs of Mdp3 were critical for its interaction with tubulin and microtubules. Cold recovery and nocodazole washout assays further demonstrated an important role for Mdp3 in regulating cellular microtubule assembly. Our data also showed that Mdp3 significantly enhanced the stability of cellular microtubules. By tubulin turbidity assay, we found that Mdp3 could promote microtubule assembly and stability in the purified system. In addition, we found that Mdp3 expression varied during the cell cycle and in primary tissues. These findings thus establish Mdp3 as a novel microtubule-binding protein that regulates microtubule assembly and stability.     

Inhibition of SNARE complex assembly differentially affects kinetic components of exocytosis

In chromaffin cells, an increase in intracellular Ca2+ leads to an exocytotic burst followed by sustained secretion. The burst can be further resolved into two kinetically distinct components, which suggests the presence of two separate pools of vesicles. To investigate how these components relate to SNARE complex formation, we introduced an antibody that blocks SNARE assembly but not disassembly. In the presence of the antibody, the sustained component was largely blocked, the burst was slightly reduced, and one of its kinetic components was eliminated. We conclude that SNARE complexes form before Ca(2+)-triggered membrane fusion and exist in a dynamic equilibrium between a loose and a tight state, both of which support exocytosis. Interaction of the antibody with preformed SNARE complexes favors the loose state.     

Membrane‐directed molecular assembly of the neuronal SNARE complex

Since the discovery and implication of N‐ethylmaleimide‐sensitive factor (NSF)‐attachment protein receptor (SNARE) proteins in membrane fusion almost two decades ago, there have been significant efforts to understand their involvement at the molecular level. In the current study, we report for the first time the molecular interaction between full‐length recombinant t‐SNAREs and v‐SNARE present in opposing liposomes, leading to the assembly of a t‐/v‐SNARE ring complex. Using high‐resolution electron microscopy, the electron density maps and 3D topography of the membrane‐directed SNARE ring complex was determined at nanometre resolution. Similar to the t‐/v‐SNARE ring complex formed when 50 nm v‐SNARE liposomes meet a t‐SNARE‐reconstituted planer membrane, SNARE rings are also formed when 50 nm diameter isolated synaptic vesicles (SVs) meet a t‐SNARE‐reconstituted planer lipid membrane. Furthermore, the mathematical prediction of the SNARE ring complex size with reasonable accuracy, and the possible mechanism of membrane‐directed t‐/v‐SNARE ring complex assembly, was determined from the study. Therefore in the present study, using both lipososome‐reconstituted recombinant t‐/v‐SNARE proteins, and native v‐SNARE present in isolated SV membrane, the membrane‐directed molecular assembly of the neuronal SNARE complex was determined for the first time and its size mathematically predicted. These results provide a new molecular understanding of the universal machinery and mechanism of membrane fusion in cells, having fundamental implications in human health and disease.     

Cysteine-disulfide cross-linking to monitor SNARE complex assembly during endoplasmic reticulum-Golgi transport

Assembly of cognate SNARE proteins into SNARE complexes is required for many intracellular membrane fusion reactions. However, the mechanisms that govern SNARE complex assembly and disassembly during fusion are not well understood. We have devised a new in vitro cross-linking assay to monitor SNARE complex assembly during fusion of endoplasmic reticulum (ER)-derived vesicles with Golgi-acceptor membranes. In Saccharomyces cerevisiae, anterograde ER-Golgi transport requires four SNARE proteins: Sec22p, Bos1p, Bet1p, and Sed5p. After tethering of ER-derived vesicles to Golgi-acceptor membranes, SNARE proteins are thought to assemble into a four-helix coiled-coil bundle analogous to the structurally characterized neuronal and endosomal SNARE complexes. Molecular modeling was used to generate a structure of the four-helix ER-Golgi SNARE complex. Based on this structure, cysteine residues were introduced into adjacent SNARE proteins such that disulfide bonds would form if assembled into a SNARE complex. Our initial studies focused on disulfide bond formation between the SNARE motifs of Bet1p and Sec22p. Expression of SNARE cysteine derivatives in the same strain produced a cross-linked heterodimer of Bet1p and Sec22p under oxidizing conditions. Moreover, this Bet1p-Sec22p heterodimer formed during in vitro transport reactions when ER-derived vesicles containing the Bet1p derivative fused with Golgi membranes containing the Sec22p derivative. Using this disulfide cross-linking assay, we show that inhibition of transport with anti-Sly1p antibodies blocked formation of the Bet1p-Sec22p heterodimer. In contrast, chelation of divalent cations did not inhibit formation of the Bet1p-Sec22p heterodimer during in vitro transport but potently inhibited Golgi-specific carbohydrate modification of glyco-pro-alpha factor. This data suggests that Ca(2+) is not directly required for membrane fusion between ER-derived vesicles and Golgi-acceptor membranes.     

Mso1 is a novel component of the yeast exocytic SNARE complex

The yeast exocytic SNARE complex consists of one molecule each of the Sso1/2 target SNAREs, Snc1/2 vesicular SNAREs, and the Sec9 target SNARE, which form a fusion complex that is conserved in evolution. Another protein, Sec1, binds to the SNARE complex to facilitate assembly. We show that Mso1, a Sec1-interacting protein, also binds to the SNARE complex and plays a role in mediating Sec1 functions. Like Sec1, Mso1 bound to SNAREs in cells containing SNARE complexes (i.e. wild-type, sec1-1, and sec18-1 cells), but not in cells in which complex formation is inhibited (i.e. sec4-8 cells). Nevertheless, Mso1 remained associated with Sec1 even in sec4-8 cells, indicating that they act as a pair. Mso1 localized primarily to the plasma membrane of the bud when SNARE complex formation was not impaired but was mostly in the cytoplasm when assembly was prevented. Genetic studies suggest that Mso1 enhances Sec1 function while attenuating Sec4 GTPase function. This dual action may impart temporal regulation between Sec4 turnoff and Sec1-mediated SNARE assembly. Notably, a small region at the C terminus of Mso1 is conserved in the mammalian Munc13/Mint proteins and is necessary for proper membrane localization. Overexpression of Mso1 lacking this domain (Mso1-(1-193)) inhibited the growth of cells bearing an attenuated Sec4 GTPase. These results suggest that Mso1 is a component of the exocytic SNARE complex and a possible ortholog of the Munc13/Mint proteins.     

GPCRs regulate the assembly of a multienzyme complex for purine biosynthesis

G protein-coupled receptors (GPCRs) transmit exogenous signals to the nucleus, promoting a myriad of biological responses via multiple signaling pathways in both healthy and cancerous cells. However, little is known about the response of cytosolic metabolic pathways to GPCR-mediated signaling. Here we applied fluorescent live-cell imaging and label-free dynamic mass redistribution assays to study whether purine metabolism is associated with GPCR signaling. Through a library screen of GPCR ligands in conjunction with live-cell imaging of a metabolic multienzyme complex for de novo purine biosynthesis, the purinosome, we demonstrated that the activation of endogenous Gα(i)-coupled receptors correlates with purinosome assembly and disassembly in native HeLa cells. Given the implications of GPCRs in mitogenic signaling and of the purinosome in controlling metabolic flux via de novo purine biosynthesis, we hypothesize that regulation of purinosome assembly and disassembly may be one of the downstream events of mitogenic GPCR signaling in human cancer cells.     

Modular complexes that regulate actin assembly in budding yeast

The actin cytoskeleton of budding yeast contains an extensive set of actin-associated proteins with conserved mammalian counterparts. For more than 20 years, yeast has been used as a model organism to dissect the in vivo functions of these factors, revealing an intricate web of genetic interactions in the cell. Now, a surge of biochemical reports is defining the physical interactions and activities of these proteins and providing mechanistic insights into their cellular roles. The emerging view is that most actin-associated proteins do not act alone but, rather, associate to form modular protein complexes that regulate actin assembly and organization.