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A new Golgi membrane enzyme that assists in the guanine nucleotide (GDP-GTP) exchange within the adenosine diphosphate-ribosylation factor has been identified through the ability of brefeldin A (BFA), a fungal metabolite, to block the exchange. BFA's inhibitory effect is strong evidence for an enzymatic nucleotide swapping device that is necessary for the development of budding vesicles.

The buttressed‐ring hypothesis, supported by recent cryo‐electron tomography analysis of docked synaptic‐like vesicles in neuroendocrine cells, postulates that prefusion SNAREpins are stabilized and organized by Synaptotagmin (Syt) ring‐like oligomers. Here, we use a reconstituted single‐vesicle fusion analysis to test the prediction that destabilizing the Syt1 oligomers destabilizes the clamp and results in spontaneous fusion in the absence of Ca2+. Vesicles in which Syt oligomerization is compromised by a ring‐destabilizing mutation dock and diffuse freely on the bilayer until they fuse spontaneously, similar to vesicles containing only v‐SNAREs. In contrast, vesicles containing wild‐type Syt are immobile as soon as they attach to the bilayer and remain frozen in place, up to at least 1 h until fusion is triggered by Ca2+. [ABSTRACT FROM AUTHOR]

Is membrane fusion an essentially passive or an active process? It could be that fusion proteins simply need to pin two bilayers together long enough, and the bilayers could do the rest spontaneously. Or, it could be that the fusion proteins play an active role after pinning two bilayers, exerting force in the bilayer in one or another way to direct the fusion process. To distinguish these alternatives, we replaced one or both of the peptidic membrane anchors of exocytic vesicle (v)-and target membrane (t)-SNAREs (soluble N-ethylmaleimide-sensitive fusion protein [NSF] attachment protein [SNAP] receptor) with covalently attached lipids. Replacing either anchor with a phospholipid prevented fusion of liposomes by the isolated SNAREs, but still allowed assembly of trans-SNARE complexes docking vesicles. This result implies an active mechanism; if fusion occurred passively, simply holding the bilayers together long enough would have been sufficient. Studies using polyisoprenoid anchors ranging from 15-55 carbons and multiple phospholipid-containing anchors reveal distinct requirements for anchors of v- and t-SNAREs to function: v-SNAREs require anchors capable of spanning both leaflets, whereas t-SNAREs do not, so long as the anchor is sufficiently hydrophobic. These data, together with previous results showing fusion is inhibited as the length of the linker connecting the helical bundle-containing rod of the SNARE complex to the anchors is increased (McNew, J.A., T. Weber, D.M. Engelman, T.H. Sollner, and J.E. Rothman. 1999. Mol. Cell. 4:415-421), suggests a model in which one activity of the SNARE complex promoting fusion is to exert force on the anchors by pulling on the linkers. This motion would lead to the simultaneous inward movement of lipids from both bilayers, and in the case of the v-SNARE, from both leaflets. Key words: lipid mixing * isoprene * liposome * lipid anchor * vesicular transport

SNARE (SNAP [soluble NSF {N-ethylmaleimide-sensitive fusion protein} attachment protein] receptor) proteins are required for many fusion processes, and recent studies of isolated SNARE proteins reveal that they are inherently capable of fusing lipid bilayers. Cis-SNARE complexes (formed when vesicle SNAREs [v-SNAREs] and target membrane SNAREs [t-SNAREs] combine in the same membrane) are disrupted by the action of the abundant cytoplasmic ATPase NSF, which is necessary to maintain a supply of uncombined v- and t-SNAREs for fusion in cells. Fusion is mediated by these same SNARE proteins, forming trans-SNARE complexes between membranes. This raises an important question: why doesn't NSF disrupt these SNARE complexes as well, preventing fusion from occurring at all? Here, we report several lines of evidence that demonstrate that SNAREpins (trans-SNARE complexes) are in fact functionally resistant to NSF, and they become so at the moment they form and commit to fusion. This elegant design allows fusion to proceed locally in the face of an overall environment that massively favors SNARE disruption. Key words: membrane fusion * SNARE * NSF * [Alpha]SNAP * liposomes

A protease-resistant core domain of the neuronal SNARE complex consists of an [Alpha]-helical bundle similar to the proposed fusogenic core of viral fusion proteins [Skehel, J. J. & Wiley, D. C. (1998) Cell 95, 871-874]. We find that the isolated core of a SNARE complex efficiently fuses artificial bilayers and does so faster than full length SNAREs. Unexpectedly, a dramatic increase in speed results from removal of the N-terminal domain of the t-SNARE syntaxin, which does not affect the rate of assembly of v-t SNARES. In the absence of this negative regulatory domain, the half-time for fusion of an entire population of lipid vesicles by isolated SNARE cores ([approximately equals] 10 min) is compatible with the kinetics of fusion in many cell types.

Regulated exocytosis, which underlies many intercellular signaling events, is a tightly controlled process often triggered by calcium ion(s) (Ca2+). Despite considerable insight into the central components involved, namely, the core fusion machinery [soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)] and the principal Ca2+ sensor [C2-domain proteins like synaptotagmin (Syt)], the molecular mechanism of Ca2+-dependent release has been unclear. Here, we report that the Ca2+-sensitive oligomers of Syt1, a conserved structural feature among several C2-domain proteins, play a critical role in orchestrating Ca2+-coupled vesicular release. This follows from pHluorin-based imaging of single-vesicle exocytosis in pheochromocytoma (PC12) cells showing that selective disruption of Syt1 oligomerization using a structure-directed mutation (F349A) dramatically increases the normally low levels of constitutive exocytosis to effectively occlude Ca2+-stimulated release. We propose a parsimonious model whereby Ca2+-sensitive oligomers of Syt (or a similar C2-domain protein) assembled at the site of docking physically block spontaneous fusion until disrupted by Ca2+. Our data further suggest Ca2+-coupled vesicular release is triggered by removal of the inhibition, rather than by direct activation of the fusion machinery. [ABSTRACT FROM AUTHOR]

Synaptotagmin‐1 (Syt1) is the primary calcium sensor (Ca2+) that mediates neurotransmitter release at the synapse. The tandem C2 domains (C2A and C2B) of Syt1 exhibit functionally critical, Ca2+‐dependent interactions with the plasma membrane. With the surface forces apparatus, we directly measure the binding energy of membrane‐anchored Syt1 to an anionic membrane and find that Syt1 binds with ~6 kBT in EGTA, ~10 kBT in Mg2+ and ~18 kBT in Ca2+. Molecular rearrangements measured during confinement are more prevalent in Ca2+ and Mg2+ and suggest that Syt1 initially binds through C2B, then reorients the C2 domains into the preferred binding configuration. These results provide energetic and mechanistic details of the Syt1 Ca2+‐activation process in synaptic transmission. [ABSTRACT FROM AUTHOR]

Neural networks are optimized to detect temporal coincidence on the millisecond timescale. Here, we offer a synthetic hypothesis based on recent structural insights into SNAREs and the C2 domain proteins to explain how synaptic transmission can keep this pace. We suggest that an outer ring of up to six curved Munc13 ' MUN' domains transiently anchored to the plasma membrane via its flanking domains surrounds a stable inner ring comprised of synaptotagmin C2 domains to serve as a work-bench on which SNAREpins are templated. This 'buttressed-ring hypothesis' affords straightforward answers to many principal and long-standing questions concerning how SNAREpins can be assembled, clamped, and then released synchronously with an action potential. [ABSTRACT FROM AUTHOR]

Golgin45 plays important roles in Golgi stack assembly and is known to bind both the Golgi stacking protein GRASP55 and Rab2 in the medial-Golgi cisternae. In this study, we sought to further characterize the cisternal adhesion complex using a proteomics approach. We report here that Acyl-CoA binding domain containing 3 ( ACBD3) is likely to be a novel binding partner of Golgin45. ACBD3 interacts with Golgin45 via its GOLD domain, while its co-expression significantly increases Golgin45 targeting to the Golgi. Furthermore, ACBD3 recruits TBC1D22, a Rab33b GTPase activating protein ( GAP), to a large multi-protein complex containing Golgin45 and GRASP55. These results suggest that ACBD3 may provide a scaffolding to organize the Golgi stacking proteins and a Rab33b- GAP at the medial-Golgi. [ABSTRACT FROM AUTHOR]

The Golgi is composed of a stack of cis, medial, trans cisternae that are biochemically distinct. The stable compartments model postulates that permanent cisternae communicate through bi-directional vesicles, while the cisternal maturation model postulates that transient cisternae biochemically mature to ensure anterograde transport. Testing either model has been constrained by the diffraction limit of light microscopy, as the cisternae are only 10–20 nm thick and closely stacked in mammalian cells. We previously described the unstacking of Golgi by the ectopic adhesion of Golgi cisternae to mitochondria. Here, we show that cargo processing and transport continue—even when individual Golgi cisternae are separated and “land-locked” between mitochondria. With the increased spatial separation of cisternae, we show using three-dimensional live imaging that cis-Golgi and trans-Golgi remain stable in their composition and size. Hence, we provide new evidence in support of the stable compartments model in mammalian cells. [ABSTRACT FROM AUTHOR]

Imaging cellular structures and organelles in living cells by long time-lapse super-resolution microscopy is challenging, as it requires dense labeling, bright and highly photostable dyes, and non-toxic conditions. We introduce a set of high-density, environment-sensitive (HIDE) membrane probes, based on the membrane-permeable silicon-rhodamine dye HMSiR, that assemble in situ and enable long time-lapse, live-cell nanoscopy of discrete cellular structures and organelles with high spatiotemporal resolution. HIDE-enabled nanoscopy movies span tens of minutes, whereas movies obtained with labeled proteins span tens of seconds. Our data reveal 2D dynamics of the mitochondria, plasma membrane and filopodia, and the 2D and 3D dynamics of the endoplasmic reticulum, in living cells. HIDE probes also facilitate acquisition of live-cell, two-color, super-resolution images, expanding the utility of nanoscopy to visualize dynamic processes and structures in living cells. [ABSTRACT FROM AUTHOR]

SNARE proteins are the core of the cell's fusion machinery and mediate virtually all known intracellular membrane fusion reactions on which exocytosis and trafficking depend. Fusion is catalyzed when vesicle-associated v-SNAREs form trans-SNARE complexes ("SNAREpins") with target membrane-associated t-SNAREs, a zippering-like process releasing ∼65 kT per SNAREpin. Fusion requires several SNAREpins, but how they cooperate is unknown and reports of the number required vary widely. To capture the collective behavior on the long timescales of fusion, we developed a highly coarse-grained model that retains key biophysical SNARE properties such as the zippering energy landscape and the surface charge distribution. In simulations the ∼65-kT zippering energy was almost entirely dissipated, with fully assembled SNARE motifs but uncomplexed linker domains. The SNAREpins self-organized into a circular cluster at the fusion site, driven by entropic forces that originate in steric-electrostatic interactions among SNAREpins and membranes. Cooperative entropic forces expanded the cluster and pulled the membranes together at the center point with high force. We find that there is no critical number of SNAREs required for fusion, but instead the fusion rate increases rapidly with the number of SNAREpins due to increasing entropic forces. We hypothesize that this principle finds physiological use to boost fusion rates to meet the demanding timescales of neurotransmission, exploiting the large number of v-SNAREs available in synaptic vesicles. Once in an unfettered cluster, we estimate ≥15 SNAREpins are required for fusion within the ∼1-ms timescale of neurotransmitter release. [ABSTRACT FROM AUTHOR]

Membrane fusion is the cell's delivery process, enabling its many compartments to receive cargo and machinery for cell growth and intercellular communication. The overall activation energy of the process must be large enough to prevent frequent and nonspecific spontaneous fusion events, yet must be low enough to allow it to be overcome upon demand by specific fusion proteins [such as soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs)]. Remarkably, to the best of our knowledge, the activation energy for spontaneous bilayer fusion has never been measured. Multiple models have been developed and refined to estimate the overall activation energy and its component parts, and they span a very broad range from 20 kBT to 150 kBT, depending on the assumptions. In this study, using a bulk lipid-mixing assay at various temperatures, we report that the activation energy of complete membrane fusion is at the lowest range of these theoretical values. Typical lipid vesicles were found to slowly and spontaneously fully fuse with activation energies of ~30 kBT. Our data demonstrate that the merging of membranes is not nearly as energy consuming as anticipated by many models and is ideally positioned to minimize spontaneous fusion while enabling rapid, SNARE-dependent fusion upon demand. [ABSTRACT FROM AUTHOR]

Synaptic soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) couple their stepwise folding to fusion of synaptic vesicles with plasma membranes. In this process, three SNAREs assemble into a stable four-helix bundle. Arguably, the first and rate-limiting step of SNARE assembly is the formation of an activated binary target (t)-SNARE complex on the target plasma membrane, which then zippers with the vesicle (v)-SNARE on the vesicle to drive membrane fusion. However, the t-SNARE complex readily misfolds, and its structure, stability, and dynamics are elusive. Using single-molecule force spectroscopy, we modeled the synaptic t-SNARE complex as a parallel three-helix bundle with a small frayed C terminus. The helical bundle sequentially folded in an N-terminal domain (NTD) and a C-terminal domain (CTD) separated by a central ionic layer, with total unfolding energy of ∼17 kBT, where kB is the Boltzmann constant and T is 300 K. Peptide binding to the CTD activated the t-SNARE complex to initiate NTD zippering with the v-SNARE, a mechanism likely shared by the mammalian uncoordinated-18-1 protein (Munc18-1). The NTD zippering then dramatically stabilized the CTD, facilitating further SNARE zippering. The subtle bidirectional t-SNARE conformational switch was mediated by the ionic layer. Thus, the t-SNARE complex acted as a switch to enable fast and controlled SNARE zippering required for synaptic vesicle fusion and neurotransmission. [ABSTRACT FROM AUTHOR]