Regulated exocytosis in neurons and neuroendocrine cells requires the formation of a stable soluble N-ethylmaleimidesensitive factor attachment protein receptor (SNARE) complex consisting of synaptobrevin-2/vesicle-associated membrane protein 2, synaptosome-associated protein of 25 kDa (SNAP-25), and syntaxin 1. This complex is subsequently disassembled by the concerted action of ␣-SNAP and the ATPases associated with different cellular activities-ATPase N-ethylmaleimide-sensitive factor (NSF). We report that NSF inhibition causes accumulation of ␣-SNAP in clusters on plasma membranes. Clustering is mediated by the binding of ␣-SNAP to uncomplexed syntaxin, because cleavage of syntaxin with botulinum neurotoxin C1 or competition by using antibodies against syntaxin SNARE motif abolishes clustering. Binding of ␣-SNAP potently inhibits Ca 2؉ -dependent exocytosis of secretory granules and SNARE-mediated liposome fusion. Membrane clustering and inhibition of both exocytosis and liposome fusion are counteracted by NSF but not when an ␣-SNAP mutant defective in NSF activation is used. We conclude that ␣-SNAP inhibits exocytosis by binding to the syntaxin SNARE motif and in turn prevents SNARE assembly, revealing an unexpected site of action for ␣-SNAP in the SNARE cycle that drives exocytotic membrane fusion. INTRODUCTIONSoluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) comprise a superfamily of small, mostly membrane-anchored proteins that mediate membrane fusion in the secretory pathway of eukaryotic cells. They are characterized by the presence of SNARE motifs, homologous stretches of 60 -70 amino acids located next to the membrane anchor domains. Key to the understanding of SNARE function in membrane fusion was the discovery of an assembly-disassembly cycle that is associated with major conformational changes. SNARE motifs of appropriate sets of SNAREs are unstructured, but they spontaneously assemble into tight complexes of extraordinary stability, forming elongated coiled-coils. When residing in different membranes, SNARE assembly leads to the formation of metastable "trans"-complexes in which the N-terminal parts of the SNARE motifs are associated, whereas the C-terminal membrane anchors are still residing in separate membranes. Progression of assembly toward the C-terminal membrane anchors is thought to proceed down a steep energy gradient and force the membranes together, resulting in fusion, with the SNAREs being converted from trans to "cis" complexes (for reviews, see Söllner, 2004;Brunger, 2005;Hong, 2005;Jahn and Scheller, 2006).To be reused in another round of fusion, SNAREs need to be reactivated by disassembly of cis-complexes, which is mediated by the hexameric ATPase N-ethylmaleimide-sensitive factor (NSF), a member of the ATPases associated with different cellular activities protein superfamily. NSF operates on all SNARE complexes and prevents accumulation of "spent" cis-complexes, thus ensuring that sufficient concentrations of free SNAREs are available for the maint...

This thesis has been written independently and with no other sources and aids than required. Marcin Barszczewski2.6.3. Plasmids 48 3. Results 49 3.1. Improved cell-free assay for exocytosis in PC12 cells 49 3.1.1. On-stage sonication of PC12 cells and stimulation of exocytosis in vitro 49 3.1.2. Imaging exocytotic release in the cell-free assay 52 3.1.3. Quantification of exocytotic activity 53 3.1.4. Application of changes in fluorescence for analysis of exocytotic events 54 3.2. Characterisation of the exocytotic responses in the improved cell-free assay 56 3.2.1. Cytosolic factors seem to modulate exocytosis differently in the presence of ATP-Mg. 57 3.2.2. Exocytosis on membrane lawns is strictly dependent on ATP-Mg. 59 3.2.3. Various ATP analogues do reduce exocytosis when introduced in two stage assay. 62 3.2.4. SNARE-specific clostridial toxin light chains reduce exocytosis in cellfree assay. 64 3.2.5. Anti SNAP-25 antibody (71.1) inhibits fusion only after extended preincubation. Soluble domains of Q-and R-SNARE proteins inhibit exocytosis on membrane lawns. 66 3.3. The α-SNAP inhibits exocytosis in the in vitro assay. 68 3.3.1. Recombinant α-SNAP blocks exocytosis which can be restored with NSF or cytosol for wild-type form of the protein but not for the dominant negative (L294A) variant. Summary 104 References 105 Curriculum vitae 121Acknowledgements 123The synaptic vesicles are loaded with neurotransmitter (1) and form a reserve (or depot) pool (2). From the reserve pool they are translocated to the proximity of plasma membrane (3), where the vesicles most likely undergo activation through many obscure processes collectively referred to as priming (4). Only the fully primed vesicles would respond rapidly to the inbound Ca 2+ and thus fuse with the plasma membrane releasing their content -the neurotransmitter molecules (5). After partial or full release of neurotransmitter 1.1.2. Translocation, tethering and docking of secretory vesicles 1.1.2.1. Translocation Mobilization of vesicles from the cytoplasm and their placement in the cortical region of the cell precedes the reactions of tethering and docking (Pfeffer, 1999). It has been postulated that ATP-Mg is required for mobilization of vesicles by ATP-Mgdependent motors such as myosin II and its Ca 2+ -regulated myosin light chain kinase.They are suspected to play a role during vesicle translocation to the cell membrane (Becker and Hart, 1999;Kumakura et al., 1994). TetheringIt is believed that the tethering and docking are influenced by proteins belonging to the family of Ras-related Rab/ypt small GPTases (or Rabs) that undergo precisely regulated cycles of re-and dephosphorylation (Geppert et al., 1997;Novick and Zerial, 1997;Sogaard et al., 1994). This feature, combined with a high specificity of Rabs for their effectors suggests, that in the active, GTP-bound form, Rab proteins may shuttle back and forth sets of proteins recruiting them to sites of fusion. Such recruitment would lead to the creation of a specific biochemical tag located on the donor...