BBA - Molecular Cell Research (v.1641, #2-3)

Long coiled-coil proteins and membrane traffic by Alison K Gillingham; Sean Munro (71-85).
Protein transport between organelles is mediated by vesicles which must accurately dock and fuse with appropriate compartments. Over the past several years a large number of long coiled-coil proteins have been identified on the Golgi and on endosomes, mostly as auto-antigens in autoimmune disorders. Based on their restricted intracellular distributions and their predicted rod-like structure, these proteins have been proposed to play a role in tethering vesicles to target organelles prior to fusion. However, such proteins may also play a structural role, for example as components of a Golgi matrix, or as scaffolds for the assembly of other factors important for fusion. This review will examine what is known about the function of these large coiled-coil proteins in membrane traffic.
Keywords: Coiled-coil; Vesicle tethering; Golgin; Golgi; Endosome; Rab GTPase;

Eukaryotic cells distribute materials among intracellular organelles and secrete into the extracellular space through cargo-loaded vesicles. A concluding step during vesicular transport is the fusion of a transport vesicle with a target membrane. SNARE proteins are essential for all vesicular fusion steps, thus they possibly comprise a conserved membrane fusion machinery. According to the “zipper” model, they assemble into stable membrane-bridging complexes that gradually bring membranes in juxtaposition. Hence, complex formation may provide the necessary energy for overcoming the repulsive forces between two membranes. During the last years, detailed structural and functional studies have extended the evidence that SNAREs are mostly in accord with the zipper model. Nevertheless, it remains unclear whether SNARE assembly between membranes directly leads to the merger of lipid bilayers.
Keywords: SNARE protein; Membrane fusion; Coiled coil;

SNARE regulators: matchmakers and matchbreakers by Jeffrey E. Gerst (99-110).
SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) are membrane-associated proteins that participate in the fusion of internal membranes in eukaryotic cells. SNAREs comprise three distinct and well-conserved families of molecules that act directly as membrane fusogens or, at the least, as elements that bring membranes into close apposition and allow for subsequent fusion events to occur. While the molecular events leading to fusion are still under debate, it is clear that a number of additional factors are required to bring about SNARE-mediated membrane fusion in vivo. Many of these factors, which collectively can be called SNARE regulators (e.g. Sec1/Munc18, synaptotagmin, GATE-16, LMA1, Munc13/UNC-13, synaptophysin, tomosyn, Vsm1, etc.), bind directly to SNAREs and are involved in the regulation of SNARE assembly as well as the ability of SNAREs to participate in trafficking events. In addition, recent studies have suggested a role for posttranslational modification (e.g., phosphorylation) in the regulation of SNARE functions. In this review the possible role of SNARE regulators in SNARE assembly and the involvement of SNARE phosphorylation in the regulation of intracellular membrane trafficking will be discussed.
Keywords: Arf-GAP; Complexin; Apg8/GATE-16; LMA1; Munc13; Munc18/Sec1; Phosphorylation; SNARE; Synaptophysin; Synaptotagmin; Tomosyn; Vsm1/Ddi1;

Control of eukaryotic membrane fusion by N-terminal domains of SNARE proteins by Lars E.P Dietrich; Christine Boeddinghaus; Tracy J LaGrassa; Christian Ungermann (111-119).
SNARE proteins function at the center of membrane fusion reactions by forming complexes with each other via their coiled-coil domains. Several SNAREs have N-terminal domains (NTDs) that precede the coiled-coil domain and have critical functions in regulating the fusion cascade. This review will highlight recent findings on NTDs of syntaxins, the longin domain of VAMP proteins and SNAP-23/25 homologues in yeast. Biochemical and genetic experiments as well as the resolution of several NMR and crystal structures of SNARE NTDs shed light on their diverse function.
Keywords: SNARE; N-terminal domain; Syntaxin; Longin; SM protein; Vam7;

Revisiting the role of SNAREs in exocytosis and membrane fusion by Joseph A. Szule; Jens R. Coorssen (121-135).
For over a decade SNARE hypotheses have been proposed to explain the mechanism of membrane fusion, yet the field still lacks sufficient evidence to conclusively identify the minimal components of native fusion. Consequently, debate concerning the postulated role(s) of SNAREs in membrane fusion continues. The focus of this review is to revisit original literature with a current perspective. Our analysis begins with the earliest studies of clostridial toxins, leading to various cellular and molecular approaches that have been used to test for the roles of SNAREs in exocytosis. We place much emphasis on distinguishing between specific effects on membrane fusion and effects on other critical steps in exocytosis. Although many systems can be used to study exocytosis, few permit selective access to specific steps in the pathway, such as membrane fusion. Thus, while SNARE proteins are essential to the physiology of exocytosis, assay limitations often prevent definitive conclusions concerning the molecular mechanism of membrane fusion. In all, the SNAREs are more likely to function upstream as modulators or priming factors of fusion.
Keywords: Secretion; Exocytosis; Clostridial toxin; Lipid; Fusion pore; Molecular mechanism;

Calcium and calmodulin in membrane fusion by Robert D. Burgoyne; Michael J. Clague (137-143).
Regulated exocytosis was the first intracellular membrane fusion step that was suggested to involve both Ca2+ and calmodulin. In recent years, it has become clear that calmodulin is not an essential Ca2+ sensor for exocytosis but that it is likely to have a more regulatory role. A requirement for cytosolic Ca2+ in other vesicle fusion events within cells has become apparent and in certain cases, such as homotypic fusion of early endosomes and yeast vacuoles, calmodulin may be the primary Ca2+ sensor. A number of distinct targets for calmodulin have been identified including SNARE proteins and subunits of the vacuolar ATPase. The extent to which calmodulin regulates different intracellular fusion events through conserved SNARE-dependent or other mechanisms remains to be resolved.
Keywords: Calcium; Calmodulin; Endocytosis; Exocytosis; Membrane traffic; Neurotransmission; SNARE; Synapse;

Involvement of LMA1 and GATE-16 family members in intracellular membrane dynamics by Zvulun Elazar; Ruth Scherz-Shouval; Hagai Shorer (145-156).
Intracellular membrane fusion is conserved from yeast to man as well as among different intracellular trafficking pathways. This process can be generally divided into several well-defined biochemical reactions. First, an early recognition (or tethering) takes place between donor and acceptor membranes, mediated by ypt/rab GTPases and complexes of tethering factors. Subsequently, a closer association between the two membranes is achieved by a docking process, which involves tight association between membrane proteins termed SNAREs. The formation of such a trans-SNARE complex leads to the final membrane fusion, resulting in an accumulation of cis-SNARE complexes on the acceptor membrane. Thus, multiple rounds of transport and delivery of the donor SNARE back to its original membrane require dissociation of the SNARE complexes. SNARE dissociation, termed priming, is mediated by the AAA ATPase, N-ethylmaleimide-sensitive factor (NSF) and its partner, soluble NSF attachment protein (SNAP), in a reaction that requires ATP hydrolysis. In the present review we focus on LMA1 and GATE-16, two low-molecular-weight proteins, which assist in priming SNARE molecules in the vacuole in yeast and the Golgi complex in mammals, respectively. LMA1 and GATE-16 are suggested to keep the dissociated cis-SNAREs apart from each other, allowing multiple fusion processes to take place. GATE-16 belongs to a novel family of ubiquitin-like proteins conserved from yeast to man. We discuss here the involvement of this family in multiple intracellular trafficking pathways.
Keywords: Membrane fusion; Golgi; Vacuole; Autophagy; SNARE; NSF; LMA1; GATE-16;

Tuning exocytosis for speed: fast and slow modes by Thomas F.J. Martin (157-165).
Exocytic fusion reactions triggered by Ca2+ are widespread in neural, endocrine, exocrine, hemapoetic and perhaps all cell types. These processes exhibit tremendous variation in latencies to fusion following a Ca2+ rise and in rates of fusion. We review reported differences for synaptic vesicle (SV) and dense-core vesicle (DCV) exocytosis and attempt to identify key features in the molecular mechanisms of docking, priming and fusion of SVs and DCVs that may account for differences in speed.
Keywords: Synaptic vesicle; Dense-core vesicle; Ca2+-dependent exocytosis; SNARE;

The fusion pore by Manfred Lindau; Guillermo Alvarez de Toledo (167-173).
The secretory process requires many different steps and stages. Vesicles must be formed and transported to the target membrane. They must be tethered or docked at the appropriate sites and must be prepared for fusion (priming). As the last step, a fusion pore is formed and the contents are released. Release of neurotransmitter is an extremely rapid event leading to rise times of the postsynaptic response of less than 100 μs. The release thus occurs during the initial formation of the exocytotic fusion pore. To understand the process of synaptic transmission, it is thus of outstanding importance to understand the molecular structure of the fusion pore, what are the properties of the initial fusion pore, how these properties affect the release process and what other factors may be limiting the kinetics of release. Here we review the techniques currently employed in fusion pore studies and discuss recent data and opinions on exocytotic fusion pore properties.
Keywords: Membrane fusion; Fusion pore; Exocytosis; Kiss-and-run; Transmitter release;

Actin and its associated proteins participate in several intracellular trafficking mechanisms. This review assesses recent work that shows how actin participates in the terminal trafficking event of membrane bilayer fusion. A recent flurry of reports defines a role for Rho proteins in membrane fusion and also demonstrates that this role is distinct from any vesicle transport mechanism. Rho proteins are well known to govern actin remodeling, which implicates this process as a condition of membrane fusion. A small but significant body of work examines actin-regulated events of intracellular membrane fusion, exocytosis and endocytosis. In general, actin has been shown to act as a negative regulator of exocytosis. Cortical actin filaments act as a barrier that requires transient removal to allow vesicles to undergo docking at the plasma membrane. However, once docked, F-actin synthesis may act as a positive regulator to give the final stimulus to drive membrane fusion. F-actin synthesis is clearly needed for endocytosis and intracellular membrane fusion events. What may seem like dissimilar results are perhaps snapshots of a single mechanism of membranous actin remodeling (i.e. dynamic disassembly and reassembly) that is universally needed for all membrane fusion events.
Keywords: Actin; Actin ligand; Rho; Membrane fusion; Exocytosis;

Work with Paramecium has contributed to the actual understanding of certain aspects of exocytosis regulation, including membrane fusion. The system is faster and more synchronous than any other dense-core vesicle system described and its highly regular design facilitates correlation of functional and ultrastructural (freeze-fracture) features. From early times on, several crucial aspects of exocytosis regulation have been found in Paramecium cells, e.g. genetically controlled microdomains (with distinct ultrastructure) for organelle docking and membrane fusion, involvement of calmodulin in establishing such microdomains, priming by ATP, occurrence of focal fusion with active participation of integral and peripheral proteins, decay of a population of integral proteins (“rosettes”, mandatory for fusion capacity) into subunits and their lateral dispersal during fusion, etc. The size of rosette particles and their dispersal upon focal fusion would be directly compatible with proteolipid V0 subunits of a V-ATPase, much better than the size predicted for oligomeric SNARE pins (SCAMPs are unknown from Paramecium at this time). However, there are some restrictions for a straightforward interpretation of ultrastructural results. The rather pointed, nipple-like tip of the trichocyst membrane could accommodate only one (or very few) potential V0 counterpart(s), while the overlaying domain of the cell membrane contains numerous rosette particles. Particle size is compatible with V0, but larger than that assumed for the SNARE complexes. When membrane fusion is induced in the presence of antibodies against cell surface components, focal fusion is seen to occur with dispersing rosette particles but without dispersal of their subunits and without pore expansion. Clearly, this is required for completing fusion and pore expansion. After cloning SNARE and V0 components in Paramecium (with increasing details becoming rapidly available), we may soon be able to address the question more directly, whether any of these components or some new ones to be detected, serve exocytotic and/or any other membrane fusions in Paramecium.
Keywords: Calcium; Exocytosis; Membrane fusion; Paramecium; Protozoa;

Mitochondrial membrane fusion by Benedikt Westermann (195-202).
Mitochondrial fusion has been observed in a great variety of organisms from yeast to man. It serves to mix and unify the mitochondrial compartment and plays roles in cellular aging, cell development, energy dissipation and mitochondrial DNA inheritance. Large GTPases in the mitochondrial outer membrane, termed Fzo or mitofusins, have been identified as key components of the mitochondrial fusion machinery in yeast, flies and mammalian cells. Recent studies in yeast suggest an involvement of a dynamin-related protein in the intermembrane space. Additional components have been identified by genetic screens. These findings suggest a unique and evolutionarily conserved mechanism for mitochondrial membrane fusion.
Keywords: Dynamin; Fzo; Membrane fusion; Mitochondrion; Mitofusin; Organelle biogenesis;