Early endosomal SNAREs form a structurally conserved SNARE complex and fuse liposomes with multiple topologies

Zwilling, Daniel; Cypionka, Anna; Pohl, Wiebke H.; Fasshauer, Dirk; Walla, Peter Jomo; Wahl, Markus C.; Jahn, Reinhard

doi:10.1038/sj.emboj.7601467

Cited by 85 publications

(116 citation statements)

References 40 publications

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“…2A) and also thoroughly depends on the specific Qabc:R topology of vacuolar SNARE RPLs (B), in accordance with previous observations (11,28). Even though this Qabc:R topological restriction is established for vacuolar SNAREs in yeast, it should be noted that the multiplicity in topologies was reported previously for mammalian early endosomal SNAREs (35).…”

Section: Resultssupporting

confidence: 90%

Distinct Contributions of Vacuolar Qabc- and R-SNARE Proteins to Membrane Fusion Specificity

Izawa

Onoue

Furukawa

et al. 2012

Journal of Biological Chemistry

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Background: Qabc-and R-SNARE proteins are key components for membrane fusion in eukaryotic organelles. Results: Reconstituted SNARE proteoliposomal study reveals distinct contributions of Qabc-and R-SNAREs to the specificity of membrane fusion. Conclusion: An assembly of proper Qabc-SNARE combinations is critical for mediating fusion specificity. Significance: This provides new insights for understanding how cells organize their complex membrane trafficking pathways.

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Section: Resultssupporting

confidence: 90%

Distinct Contributions of Vacuolar Qabc- and R-SNARE Proteins to Membrane Fusion Specificity

Izawa

Onoue

Furukawa

et al. 2012

Journal of Biological Chemistry

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“…When these liposomes are mixed, fusion is rapid, with the stabilizing R-SNARE fragment being displaced during the reaction (23,24). Liposomes were labeled with Oregon Green (usually containing a stabilized acceptor complex) or with Texas Red, resulting in robust FRET upon fusion (25). Lipid mixing is commonly used as a read-out for fusion, despite some caveats (26).…”

Section: Resultsmentioning

confidence: 99%

“…To distinguish the population of fused from docked liposomes, we simultaneously measured lipid mixing by the decrease of the fluorescence lifetime of the donor dye as an accurate read-out for FRET (Fig. S2) (25). In contrast to the cross-correlation, no changes in the fluorescence lifetime were observed immediately after mixing (black and green curve in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The fluorescence time traces were analyzed for individual fluorescence bursts or using fluorescence auto-and cross-correlation spectroscopy (FCS and FCCS) (25,34,35) in combination with fluorescence lifetime analysis. FCS allows for the determination of the average number of red or green labeled particles being in the focal detection volume by analyzing signal fluctuations in the red or green detector that are caused by the diffusion of the particles in an out the detection volume (36) (see SI Appendix).…”

Section: Methodsmentioning

confidence: 99%

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Discrimination between docking and fusion of liposomes reconstituted with neuronal SNARE-proteins using FCS

Cypionka

Stein

Hernandez

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Neuronal exocytosis is mediated by the SNARE proteins synaptobrevin 2/VAMP, syntaxin 1A, and SNAP-25A. While it is wellestablished that these proteins mediate membrane fusion after reconstitution in artificial membranes, it has so far been difficult to monitor intermediate stages of the reaction. Using a confocal two-photon setup, we applied fluorescence cross-correlation spectroscopy (FCCS) and fluorescence lifetime analysis to discriminate between docking and fusion of liposomes. We show that liposome populations that are either non-interacting, or are undergoing docking and fusion, as well as multiple interactions can be quantitatively discriminated without the need for immobilizing the lipid bilayers. When liposomes containing a stabilized syntaxin 1A/ SNAP-25A complex were mixed with liposomes containing synaptobrevin 2, we observed that rapid docking precedes fusion. Accordingly, docked intermediates accumulated in the initial phase of the reaction. Furthermore, rapid formation of multiple docked states was observed with on average four liposomes interacting with each other. When liposomes of different sizes were compared, only the rate of lipid mixing depended on the liposome size but not the rate of docking. Our results show that under appropriate conditions a docked state, mediated by trans-SNARE interactions, can be isolated that constitutes an intermediate in the fusion pathway.fluorescence cross-correlation spectroscopy ͉ fluorescence lifetime analysis ͉ fusion intermediate ͉ single-particle detection ͉ SNAREs

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“…In addition to its lower T m , the S. cerevisiae SNARE complex is not SDS-resistant. The early and late endosomal SNARE complexes have melting temperatures of 87 (12) and 78°C, respectively (20).…”

Section: Resultsmentioning

confidence: 99%

The Structure of the Yeast Plasma Membrane SNARE Complex Reveals Destabilizing Water-filled Cavities

Strop

Kaiser

Vrljic

et al. 2008

Journal of Biological Chemistry

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SNARE proteins form a complex that leads to membrane fusion between vesicles, organelles, and plasma membrane in all eukaryotic cells. We report the 1.7 Å resolution structure of the SNARE complex that mediates exocytosis at the plasma membrane in the yeast Saccharomyces cerevisiae. Similar to its neuronal and endosomal homologues, the S. cerevisiae SNARE complex forms a parallel four-helix bundle in the center of which is an ionic layer. The S. cerevisiae SNARE complex exhibits increased helix bending near the ionic layer, contains waterfilled cavities in the complex core, and exhibits reduced thermal stability relative to mammalian SNARE complexes. Mutagenesis experiments suggest that the water-filled cavities contribute to the lower stability of the S. cerevisiae complex.Membrane fusion is a fundamental and highly regulated process that is required for the transport of proteins, lipids, and metabolites in all eukaryotes. Highly conserved SNARE 2 (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins play a key role in the fusion of a transport vesicle with its target membrane (1, 2). Specific sets of SNAREs located on vesicle and target membranes form a complex that draws together SNARE transmembrane domains, leading to juxtaposition and fusion of the two lipid membranes. SNARE-mediated fusion processes are either constitutive or triggered, and they require additional SNARE-interacting factors, such as Munc13, Sec1/Munc 18-like proteins, synaptotagmins, and complexins (3, 4).Neuronal SNAREs play a key role in the fusion of synaptic vesicles with the plasma membrane, a process that is critical for neurotransmission (5). Synaptic vesicles dock at the plasma membrane and upon cell depolarization and Ca 2ϩ entry fuse with the plasma membrane, thus releasing neurotransmitters into the synaptic cleft. The neuronal SNARE complex is composed of synaptobrevin 2, which is primarily localized to synaptic vesicles, and syntaxin 1A and SNAP-25, which are primarily associated with the plasma membrane.The family of yeast SNAREs mediates constitutive fusion between transport vesicles and intracellular organelles or the plasma membrane (6, 7). Yeast has been a valuable system for studying membrane fusion and vesicular transport because of the ease of genetic and biochemical manipulations. Yeast is also one of the first organisms that evolved to utilize intracellular membrane fusion and therefore provides a valuable snapshot of the fusion machinery in lower eukaryotes. The Saccharomyces cerevisiae SNARE proteins involved in exocytosis at the plasma membrane (the synaptobrevin homologue Snc1p, the syntaxin homologue Sso1p, and the SNAP-25 homologue Sec9p) show assembly properties similar to their neuronal and endosomal homologues. Relative to the neuronal SNARE complex the S. cerevisiae SNARE complex exhibits decreased thermal stability, and prior to SNARE complex formation, Sso1p interacts with Sec9p with 1:1 stoichiometry (as opposed to a mixture of 1:1 and 1:2 for the neuronal SNAREs) (8, 9).Several...

show abstract

Early endosomal SNAREs form a structurally conserved SNARE complex and fuse liposomes with multiple topologies

Cited by 85 publications

References 40 publications

Distinct Contributions of Vacuolar Qabc- and R-SNARE Proteins to Membrane Fusion Specificity

Distinct Contributions of Vacuolar Qabc- and R-SNARE Proteins to Membrane Fusion Specificity

Discrimination between docking and fusion of liposomes reconstituted with neuronal SNARE-proteins using FCS

The Structure of the Yeast Plasma Membrane SNARE Complex Reveals Destabilizing Water-filled Cavities

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