Ca2+-sensor synaptotagmin-1 is thought to trigger membrane fusion by binding to acidic membrane lipids and SNARE proteins. Previous work has shown that binding is mediated by electrostatic interactions that are sensitive to the ionic environment. However, the influence of divalent or polyvalent ions, at physiological concentrations, on synaptotagmin binding to membranes or SNAREs has not been explored. Here we show that binding of rat synaptotagmin-1 to membranes containing PIP2 is regulated by charge shielding caused by the presence of divalent cations. Surprisingly, polyvalent ions such as ATP and Mg2+ completely abrogate synaptotagmin-1 binding to SNAREs regardless of whether Ca2+ is present or not. Altogether, our data suggest that at physiological ion concentrations Ca2+-dependent synaptotagmin-1 binding is confined to PIP2-containing membrane patches in the plasma membrane, suggesting that membrane interaction of synaptotagmin-1 rather than SNARE binding triggers exocytosis of vesicles.

Background: Soluble N-ethylmaleimide-sensitive factor attachment protein ␣ (␣-SNAP) regulates the pre-fusion step as well as SNARE disassembly. Results: ␣-SNAP on its own interferes with SNARE zippering and inhibits chromaffin granule fusion, but not synaptic vesicle fusion. Conclusion: Retardation of SNARE zippering by ␣-SNAP results in the partial SNARE zippering. Significance: This is the first direct evidence showing the partial SNARE zippering in the physiological context.

Soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins mediate intracellular membrane fusion in the secretory pathway. They contain conserved regions, termed SNARE motifs, that assemble between opposing membranes directionally from their N termini to their membrane-proximal C termini in a highly exergonic reaction. However, how this energy is utilized to overcome the energy barriers along the fusion pathway is still under debate. Here, we have used mutants of the SNARE synaptobrevin to arrest trans-SNARE zippering at defined stages. We have uncovered two distinct vesicle docking intermediates where the membranes are loosely and tightly connected, respectively. The tightly connected state is irreversible and independent of maintaining assembled SNARE complexes. Together, our results shed new light on the intermediate stages along the pathway of membrane fusion.

Legionella pneumophila, the Gram-negative pathogen causing Legionnaires’ disease, infects host cells by hijacking endocytic pathways and forming a Legionella-containing vacuole (LCV) in which the bacteria replicate. To promote LCV expansion and prevent lysosomal targeting, effector proteins are translocated into the host cell where they alter membrane traffic. Here we show that three of these effectors [LegC2 (Legionella eukaryotic-like gene C2)/YlfB (yeast lethal factor B), LegC3, and LegC7/YlfA] functionally mimic glutamine (Q)-SNARE proteins. In infected cells, the three proteins selectively form complexes with the endosomal arginine (R)-SNARE vesicle-associated membrane protein 4 (VAMP4). When reconstituted in proteoliposomes, these proteins avidly fuse with liposomes containing VAMP4, resulting in a stable complex with properties resembling canonical SNARE complexes. Intriguingly, however, the LegC/SNARE hybrid complex cannot be disassembled by N-ethylmaleimide-sensitive factor. We conclude that LegCs use SNARE mimicry to divert VAMP4-containing vesicles for fusion with the LCV, thus promoting its expansion. In addition, the LegC/VAMP4 complex avoids the host’s disassembly machinery, thus effectively trapping VAMP4 in an inactive state.

We have studied the adhesion state (also denoted by docking state) of lipid vesicles as induced by the divalent ions Ca or Mg at well-controlled ion concentration, lipid composition, and charge density. The bilayer structure and the interbilayer distance in the docking state were analyzed by small-angle x-ray scattering. A strong adhesion state was observed for DOPC:DOPS vesicles, indicating like-charge attraction resulting from ion correlations. The observed interbilayer separations of ∼1.6 nm agree quantitatively with the predictions of electrostatics in the strong coupling regime. Although this phenomenon was observed when mixing anionic and zwitterionic (or neutral) lipids, pure anionic membranes (DOPS) with highest charge density σ resulted in a direct phase transition to a multilamellar state, which must be accompanied by rupture and fusion of vesicles. To extend the structural assay toward protein-controlled docking and fusion, we have characterized reconstituted N-ethylmaleimide-sensitive factor attachment protein receptors in controlled proteoliposome suspensions by small-angle x-ray scattering.

Intrinsically disordered proteins (IDPs) and their conformational transitions play an important role in neurotransmitter release at the neuronal synapse. Here, the SNARE proteins are essential by forming the SNARE complex that drives vesicular membrane fusion. While it is widely accepted that the SNARE proteins are intrinsically disordered in their monomeric prefusion form, important mechanistic aspects of this prefusion conformation and its lipid interactions, before forming the SNARE complex, are not fully understood at the molecular level and remain controversial. Here, by a combination of NMR and fluorescence spectroscopy methods, we find that vesicular synaptobrevin-2 (syb-2) in its monomeric prefusion conformation shows high flexibility, characteristic for an IDP, but also a high dynamic range and increasing rigidity from the N to C terminus. The gradual increase in rigidity correlates with an increase in lipid binding affinity from the N to C terminus. It could also explain the increased rate for C-terminal SNARE zippering, known to be faster than N-terminal SNARE zippering. Also, the syb-2 SNARE motif and, in particular, the linker domain show transient and weak membrane binding, characterized by a high off-rate and low (millimolar) affinity. The transient membrane binding of syb-2 may compensate for the repulsive forces between the two membranes and/or the SNARE motifs and the membranes, helping to destabilize the hydrophilic-hydrophobic boundary in the bilayer. Therefore, we propose that optimum flexibility and membrane binding of syb-2 regulate SNARE assembly and minimize repulsive forces during membrane fusion.

My special thanks go to Nickias for his endless love and support. I would be lost many times without him being on the other side of the line. Every time an experiment put me up or down, he invented a new way to reset 700 kilometers to zero. Vielen Dank, Nicki! These moments were only a little part of what we have shared and will ever do happily.The last but not the least, I am very indebted to my family and would like to dedicate this thesis to them. Sizin desteğiniz olmadan bu sayfaların hiç biri yazılamazdı. Bilim merakıma benden çok sahip çıktığınız için, her aylığınızın ilk kısmını kitapçıda harcadığımız için teşekkür ederim. Beni beni bu denli açık fikirli, mutlu yetiştiren her fedakarlığınıza binlerce kez minnetarım. Tezimi anneme, babama ve biricik kız kardeşime armağan ediyorum.

The approximately 350 ion channels encoded by the mammalian genome are a main pillar of the nervous system. We have determined the expression pattern of 320 channels in the two-week-old (P14) rat brain by means of non-radioactive robotic in situ hybridization. Optimized methods were developed and implemented to generate stringently coronal brain sections. The use of standardized methods permits a direct comparison of expression patterns across the entire ion channel expression pattern data set and facilitates recognizing ion channel co-expression. All expression data are made publically available at the Genepaint.org database. Inwardly rectifying potassium channels (Kir, encoded by the Kcnj genes) regulate a broad spectrum of physiological processes. Kcnj channel expression patterns generated in the present study were fitted with a deformable subdivision mesh atlas produced for the P14 rat brain. This co-registration, when combined with numerical quantification of expression strengths, allowed for semi-quantitative automated annotation of expression patterns as well as comparisons among and between Kcnj subfamilies. The expression patterns of Kcnj channel were also cross validated against previously published expression patterns of Kcnj channel genes.Electronic supplementary materialThe online version of this article (doi:10.1007/s12021-014-9247-0) contains supplementary material, which is available to authorized users.