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Single vesicle millisecond fusion kinetics reveals number of SNARE complexes optimal for fast SNARE-mediated membrane fusion.

ABSTRACT: SNAREs mediate membrane fusion in intracellular vesicle traffic and neuronal exocytosis. Reconstitution of membrane fusion in vitro proved that SNAREs constitute the minimal fusion machinery. However, the slow fusion rates observed in these systems are incompatible with those required in neurotransmission. Here we present a single vesicle fusion assay that records individual SNARE-mediated fusion events with millisecond time resolution. Docking and fusion of reconstituted synaptobrevin vesicles to target SNARE complex-containing planar membranes are distinguished by total internal reflection fluorescence microscopy as separate events. Docking and fusion are SNAP-25-dependent, require no Ca(2+), and are efficient at room temperature. Analysis of the stochastic data with sequential and parallel multi-particle activation models reveals six to nine fast-activating steps. Of all the tested models, the kinetic model consisting of eight parallel reaction rates statistically fits the data best. This might be interpreted by fusion sites consisting of eight SNARE complexes that each activate in a single rate-limiting step in 8 ms.

SUBMITTER: Domanska MK

PROVIDER: S-EPMC2797286 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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Single vesicle millisecond fusion kinetics reveals number of SNARE complexes optimal for fast SNARE-mediated membrane fusion.

Domanska Marta K MK Kiessling Volker V Stein Alexander A Fasshauer Dirk D Tamm Lukas K LK

The Journal of biological chemistry 20090915 46

SNAREs mediate membrane fusion in intracellular vesicle traffic and neuronal exocytosis. Reconstitution of membrane fusion in vitro proved that SNAREs constitute the minimal fusion machinery. However, the slow fusion rates observed in these systems are incompatible with those required in neurotransmission. Here we present a single vesicle fusion assay that records individual SNARE-mediated fusion events with millisecond time resolution. Docking and fusion of reconstituted synaptobrevin vesicles ...[more]

PMID: 19759010

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Project description:Protein synthesis begins at free ribosomes or ribosomes attached with the endoplasmic reticulum (ER). Newly synthesized proteins are transported to the plasma membrane for secretion through conventional or unconventional pathways. In conventional protein secretion, proteins are transported from the ER lumen to Golgi lumen and through various other compartments to be secreted at the plasma membrane, while unconventional protein secretion bypasses the Golgi apparatus. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins are involved in cargo vesicle trafficking and membrane fusion. The ER localized vesicle associated SNARE (v-SNARE) protein Sec22 plays a major role during anterograde and retrograde transport by promoting efficient membrane fusion and assisting in the assembly of higher order complexes by homodimer formation. Sec22 is not only confined to ER-Golgi intermediate compartments (ERGIC) but also facilitates formation of contact sites between ER and plasma membranes. Sec22 mutation is responsible for the development of atherosclerosis and symptoms in the brain in Alzheimer's disease and aging in humans. In the fruit fly Drosophila melanogaster, Sec22 is essential for photoreceptor morphogenesis, the wingless signaling pathway, and normal ER, Golgi, and endosome morphology. In the plant Arabidopsis thaliana, it is involved in development, and in the nematode Caenorhabditis elegans, it is in involved in the RNA interference (RNAi) pathway. In filamentous fungi, it affects cell wall integrity, growth, reproduction, pathogenicity, regulation of reactive oxygen species (ROS), expression of extracellular enzymes, and transcriptional regulation of many development related genes. This review provides a detailed account of Sec22 function, summarizes its domain structure, discusses its genetic redundancy with Ykt6, discusses what is known about its localization to discrete membranes, its contributions in conventional and unconventional autophagy, and a variety of other roles across different cellular systems ranging from higher to lower eukaryotes, and highlights some of the surprises that have originated from research on Sec22.

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Single vesicle millisecond fusion kinetics reveals number of SNARE complexes optimal for fast SNARE-mediated membrane fusion.

Publications

Single vesicle millisecond fusion kinetics reveals number of SNARE complexes optimal for fast SNARE-mediated membrane fusion.

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