SNAREs and SNARE regulators in membrane fusion and exocytosis

Eukaryotes have a remarkably well-conserved apparatus for the trafficking of proteins between intracellular compartments and delivery to their target organelles. This apparatus comprises the secretory (or ‘protein export’) pathway, which is responsible for the proper processing and delivery of proteins and lipids, and is essential for the derivation and maintenance of those organelles. Protein transport between intracellular compartments is mediated by carrier vesicles that bud from one organelle and fuse selectively with another. Therefore, organelle-specific trafficking of vesicles requires specialized proteins that regulate vesicle transport, docking and fusion. These proteins are generically termed SNAREs and comprise evolutionarily conserved families of membrane-associated proteins (i.e. the synaptobrevin/VAMP, syntaxin and SNAP-25 families) which mediate membrane fusion. SNAREs act at all levels of the secretory pathway, but individual family members tend to be compartment-specific and, thus, are thought to contribute to the specificity of docking and fusion events. In this review, we describe the different SNARE families which function in exocytosis, as well as discuss the role of possible negative regulators (e.g. ‘SNARE-masters’) in mediating events leading to membrane fusion. A model to illustrate the dynamic cycling of SNAREs between fusion-incompetent and fusion-competent states, called the SNARE cycle, is presented. Received 8 October 1998; received after revision 26 November 1998; accepted 26 November 1998     

Membrane traffic in the secretory pathway

Secretion is a fundamental biological activity of all eukaryotic cells by which they release certain substances in the extracellular space. It is considered a specialized mode of membrane trafficking that is achieved by docking and fusion of secretory vesicles to the plasma membrane (i.e., exocytosis). Secretory vesicle traffic is thought to be regulated by a family of Rab small GTPases, which are regulators of membrane traffic that are common to all eukaryotic cells. Classically, mammalian Rab3 subfamily members were thought to be critical regulators of secretory vesicle exocytosis in neurons and endocrine cells, but recent genetic and proteomic studies indicate that Rab3 is not the sole Rab isoform that regulates secretory vesicle traffic. Rather, additional Rab isoforms, especially Rab27 subfamily members, are required for this process. In this article I review the current literature on the function of Rab isoforms and their effectors in regulated secretory vesicle traffic.     

Putting a brake on synaptic vesicle endocytosis

In chemical synapses, action potentials evoke synaptic vesicle fusion with the presynaptic membrane at the active zone to release neurotransmitter. Synaptic vesicle endocytosis (SVE) then follows exocytosis to recapture vesicle proteins and lipid components for recycling and the maintenance of membrane homeostasis. Therefore, SVE plays an essential role during neurotransmission and is one of the most precisely regulated biological processes. Four modes of SVE have been characterized and both positive and negative regulators have been identified. However, our understanding of SVE regulation remains unclear, especially the identity of negative regulators and their mechanisms of action. Here, we review the current knowledge of proteins that function as inhibitors of SVE and their modes of action in different forms of endocytosis. We also propose possible physiological roles of such negative regulation. We believe that a better understanding of SVE regulation, especially the inhibitory mechanisms, will shed light on neurotransmission in health and disease.     

Preparing the lethal hit: interplay between exo- and endocytic pathways in cytotoxic T lymphocytes

Cytotoxic T lymphocytes patrol our body in search for infected cells which they kill through the release of cytotoxic substances contained in cytotoxic granules. The fusion of cytotoxic granules occurs at a specially formed contact site, the immunological synapse, and is tightly controlled to ensure specificity. In this review, we discuss the contribution of two intracellular compartments, endosomes and cytotoxic granules, to the formation, function and disassembly of the immunological synapse. We highlight a recently proposed sequential process of fusion events at the IS upon target cell recognition. First, recycling endosomes fuse with the plasma membrane to deliver cargo required for the docking of cytotoxic granules. Second, cytotoxic granules arrive and fuse upon docking in a SNARE-dependent manner. Following fusion, membrane components of the cytotoxic granule are retrieved through endocytosis to ensure the fast, efficient serial killing of target cells that is characteristic of cytotoxic T lymphocytes.     

Membrane fusion: the process and its energy suppliers

Membrane fusion constitutes a pivotal process in eukaryotic cell physiology. Both specialized proteins and membrane lipids play key roles in fusion. Here, our current understanding of the mechanism of membrane fusion is reviewed. The focus is on the relatively simple and well-understood proteinaceous fusion machinery of enveloped viruses and the physical properties of lipids that appear to be of great relevance for fusion progression. Recent observations suggest that viral fusion proteins use packed conformational energy and bilayer-destabilizing domains to (i) bring participating membranes into intimate contact, (ii) merge proximal lipid monolayers through highly curved stalk/hemifusion intermediates, and (iii) generate a lipid-containing fusion pore, thereby terminating the fusion process. Received 4 January 2002; received after revision 3 April 2002; accepted 5 April 2002     

Population dynamics of soil inhabiting mites in cucumber treated with granular insect growth regulators (IGRs).

In small field trials, certain granular insect growth regulators (IGRs) induced significant reduction in soil inhabiting mites of cucumber plantation at a rate as low as 0.25 kg a.i./acre. However, suborders specificity relationship could easily be detected.     

The SARS-CoV S glycoprotein

The severe acute respiratory syndrome-coronavirus (SARS-CoV) spike (S) glycoprotein alone can mediate the membrane fusion required for virus entry and cell fusion. It is also a major immunogen and a target for entry inhibitors. Recent rapid advances in our knowledge of the structure and function of this protein have lead to the development of a number of candidate vaccine immunogens and SARS-CoV entry inhibitors.     

Induction of dolykaryocytes by vesicular stomatitis virus in XC rat cells]

V.S.V. induced polycaryocytes in rat embryonic fibroblasts, transformed by the Prague strain of Sarcoma Rous (XC cells). This fusion is strictly dependent on the expression of the viral genome and is probably due to the incorporation of viral antigens in the cell membrane. The integrity of cellular RNA synthesis is however not required. The fusion is probably due to a membrane structure characteristic of these transformed cells.     

Membrane fusion

The factors involved in the regulation of biological membrane fusion and models proposed for the molecular mechanism of biomembrane fusion are reviewed. The results obtained in model systems are critically discussed in the light of the known properties of biomembranes and characteristics of biomembrane fusion. Biological membrane fusion is a local-point event; extremely fast, non-leaky, and under strict control. Fusion follows on a local and most probably protein-modulated destabilization, and a transition of the interacting membranes from a bilayer to a non-bilayer lipid structure. The potential role of type II non-bilayer preferring lipids and of proteins in the local destabilization of the membranes is evaluated. Proteins are not only responsible for the mutual recognition of the fusion partners, but are most likely also to be involved in the initiation of biomembrane fusion, by locally producing or activating fusogens, or by acting as fusogens.     

Membrane fusion

Summary The factors involved in the regulation of biological membrane fusion and models proposed for the molecular mechanism of biomembrane fusion are reviewed. The results obtained in model systems are critically discussed in the light of the known properties of biomembranes and characteristics of biomembrane fusion. Biological membrane fusion is a local-point event; extremely fast, non-leaky, and under strict control. Fusion follows on a local and most probably protein-modulated destabilization, and a transition of the interacting membranes from a bilayer to a non-bilayer lipid structure. The potential role of type II non-bilayer preferring lipids and of proteins in the local destabilization of the membranes is evaluated. Proteins are not only responsible for the mutual recognition of the fusion partners, but are most likely also to be involved in the initiation of biomembrane fusion, by locally producing or activating fusogens, or by acting as fusogens.     

Structures of vesicular stomatitis virus glycoprotein: membrane fusion revisited

Glycoprotein G of the vesicular stomatitis virus (VSV) is involved in receptor recognition at the host cell surface and then, after endocytosis of the virion, triggers membrane fusion via a low pH-induced structural rearrangement. G is an atypical fusion protein, as there is a pH-dependent equilibrium between its pre- and post-fusion conformations. The atomic structures of these two conformations reveal that it is homologous to glycoprotein gB of herpesviruses and that it combines features of the previously characterized class I and class II fusion proteins. Comparison of the structures of G pre- and postfusion states shows a dramatic reorganization of the molecule that is reminiscent of that of paramyxovirus fusion protein F. It also allows identification of conserved key residues that constitute pH-sensitive molecular switches. Besides the similarities with other viral fusion machineries, the fusion properties and structures of G also reveal some striking particularities that invite us to reconsider a few dogmas concerning fusion proteins.     

The ancient claudin Dni2 facilitates yeast cell fusion by compartmentalizing Dni1 into a membrane subdomain

Dni1 and Dni2 facilitate cell fusion during mating. Here, we show that these proteins are interdependent for their localization in a plasma membrane subdomain, which we have termed the mating fusion domain. Dni1 compartmentation in the domain is required for cell fusion. The contribution of actin, sterol-dependent membrane organization, and Dni2 to this compartmentation was analysed, and the results showed that Dni2 plays the most relevant role in the process. In turn, the Dni2 exit from the endoplasmic reticulum depends on Dni1. These proteins share the presence of a cysteine motif in their first extracellular loop related to the claudin GLWxxC(8–10 aa)C signature motif. Structure–function analyses show that mutating each Dni1 conserved cysteine has mild effects, and that only simultaneous elimination of several cysteines leads to a mating defect. On the contrary, eliminating each single cysteine and the C-terminal tail in Dni2 abrogates Dni1 compartmentation and cell fusion. Sequence alignments show that claudin trans-membrane helixes bear small-XXX-small motifs at conserved positions. The fourth Dni2 trans-membrane helix tends to form homo-oligomers in Escherichia plasma membrane, and two concatenated small-XXX-small motifs are required for efficient oligomerization and for Dni2 export from the yeast endoplasmic reticulum. Together, our results strongly suggest that Dni2 is an ancient claudin that blocks Dni1 diffusion from the intercellular region where two plasma membranes are in close proximity, and that this function is required for Dni1 to facilitate cell fusion.     

An increase in Sendai virus-induced cell fusion of erythrocytes infected with Plasmodium chabaudi

The extent of cell fusion induced by Sendai virus was examined in erythrocytes infected with Plasmodium chabaudi. An increase in cell fusion of erythrocytes with Ehrlich tumor cells and of erythrocytes with erythrocytes was observed with the infected erythrocytes. However, agglutination by the virus was not changed between erythrocytes of normal and malarial mice. These results indicate that the increase in cell fusion occurred in the process of membrane fusion, suggesting that some membrane property of Plasmodium-parasitized erythrocytes is changed in terms of Sendai virus-induced cell fusion.     

Regulatory mechanisms of mitogen-activated kinase signaling

MAP kinases (MAPKs) are evolutionarily conserved regulators that mediate signal transduction and play essential roles in various physiological processes. There are three main families of MAPKs in mammals, whose functions are regulated by activators, inactivators, substrates and scaffolds, which together form delicate signaling cascades in response to different extracellular or intracellular stimulation. MAPK signaling is tightly regulated so that optimal biological activities are achieved and health is maintained. However, how the specificity of the signaling flow along each cascade is achieved is still relatively unclear. In this review, we summarize recent advances in understanding the regulation of MAPK cascades and the roles of MAP kinases and their regulators in development and in immune responses. Received 11 January 2007; received after revision 31 May 2007; accepted 5 July 2007     

The role of transmembrane domains in membrane fusion

Biological membrane fusion is driven by different types of molecular fusion machines. Most of these proteins are membrane-anchored by single transmembrane domains. SNARE proteins are essential for intracellular membrane fusion along the secretory and endocytic pathway, while various viral fusogens mediate infection of eukaryotic cells by enveloped viruses. Although both types of fusion proteins are evolutionarily quite distant from each other, they do share a number of structural and functional features. Their transmembrane domains are now known to be critical for the fusion reaction. We discuss at which stages they might contribute to bilayer mixing. Received 5 October 2006; received after revision 14 November 2006; accepted 8 January 2007     

Regulation of SNARE fusion machinery by fatty acids

Vesicle fusion is a ubiquitous biological process involved in membrane trafficking and a variety of specialised events such as exocytosis and neurite outgrowth. The energy to drive biological membrane fusion is provided by fusion proteins called SNAREs. Indeed, SNARE proteins play critical roles in neuronal development as well as neurotransmitter and hormone release. SNARE proteins form a very tight alpha-helical bundle that can pull two membranes together, thereby initiating fusion. Whereas a great deal of attention has been paid to partner proteins that can affect SNARE function, recent genetic and biochemical evidence suggests that local lipid environment may be as important in SNARE regulation. Direct lipid modification of SNARE fusion proteins and their regulation by fatty acids following phospholipase action will be discussed here in detail. Our analysis highlights the fact that lipids are not a passive platform in vesicle fusion but intimately regulate SNARE function. Received 20 December 2006; received after revision 6 February 2007; accepted 15 March 2007     

The molecular machinery of synaptic vesicle exocytosis

At the synapse, neurotransmitters are released via Ca(2+)-triggered exocytotic fusion of synaptic vesicles with the presynaptic plasma membrane. Synaptic vesicle exocytosis seems to share many basic principles and homologous proteins with other membrane fusion events. Conserved components of the general fusion machinery that participate in synaptic vesicle exocytosis include soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), ATPase N-ethylmaleimide-sensitive factor, Munc18/nSec1, Rab3 GTPase, and the exocyst proteins. In addition, synaptic vesicle exocytosis uses a set of unique components, such as synaptotagmin, complexin, Munc13, and RIM, to meet the special needs of fast Ca(2+)-triggered neurotransmitter release. This review summarizes present knowledge about the molecular mechanisms by which these components mediate and/or regulate synaptic vesicle exocytosis.     

Extracellular matrix components in intestinal development

Intestinal morphogenesis and differentiation are dependent on heterotypic cell interactions between embryonic epithelial cells (endoderm) and stromal cells (mesenchyme). Extracellular matrix molecules represent attractive candidates for regulators of these interactions. The structural and functional diversity of the extracellular matrix as intestinal development proceeds is demonstrated by 1) spatio-temporal specific expression of the classically described constituents, 2) the finding of laminin and collagen IV variants, 3) changes in the ratio of individual constituent chains, and 4) a stage-specific regulation of basement membrane molecule production, in particular by glucocorticoids. The orientation/assembly of these extracellular matrix molecules could direct precise cellular functions through interactions via integrin molecules. The involvement of extracellular matrix, and in particular basement membrane molecules in heterotypic cell interactions leading to epithelial cell differentiation, has been highlighted by the use of experimental models such as cocultures, hybrid intestines and antisense approaches. These models allowed us to conclude that a correct elaboration and assembly of the basement membrane, following close contacts between epithelial and fibroblastic cells, is necessary for the expression of differentiation markers such as digestive enzymes.     

Introduction: lipids as regulators of cell function

It is becoming increasingly clear that lipids are key regulators of cellular function and that these effects are quite diverse. First, the lipid environment in the cellular membrane bilayer is important in maintaining the normal function of receptors, enzymes, transporters and so on that are localized in the membrane. Phosphoinositides are important regulators of signalling molecules. Lipid metabolites formed by a number of enzymes including the cyclooxygenases, lipoxygenases and P450s also mediate important cellular functions. Fatty acids and lipid metabolites can also activate the nuclear peroxisome proliferator-activated receptors. Finally, a wide variety of lipid molecules are generated nonenzymatically by free-radical mechanisms that also exert potent biological effects in a wide variety of organs. Presented are a series of eight reviews that broadly cover all of these topics in some detail.     

An increase in Sendai virus-induced cell fusion of erythrocytes infected withPlasmodium chabaudi

Summary The extent of cell fusion induced by Sendai virus was examined in erythrocytes infected withPlasmodium chabaudi. An increase in cell fusion of erythrocytes with Ehrlich tumor cells and of erythrocytes with erythrocytes was observed wit the infected erythrocytes. However, agglutination by the virus was not changed between erythrocytes of normal and malarial mice. These results indicate that the increase in cell fusion occurred in the process of membrane fusion, suggesting that some membrane property ofPlasmodium-parasitized erythrocytes is changed in terms of Sendai virus-induced cell fusion.We thank Drs George L. Gerton, T. Matsuyama and M. Niwa for their comments on this work and Mr I. Kimata for preparing photographs.