Porosomes are the universal secretory portals at the cell plasma membrane, where membrane-bound secretory vesicles transiently dock and fuse to expel intravesicular contents to the outside during cell secretion. In the past decade, the neuronal porosome complex, a 10–15 nm cup-shaped lipoprotein structure has been isolated, its partial composition and 3D contour map determined, and it has been functionally reconstituted into artificial lipid membrane. Here we further determine the composition of the neuronal porosome proteome using immunoisolation and gel filtration chromatography, followed by tandem mass spectrometry. Results from the study demonstrate nearly 40 proteins to constitute the neuronal porosome proteome. Furthermore, interaction of proteins within the porosome and their resulting arrangement is predicted. The association and dissociation of proteins at the porosome following stimulation of cell secretion demonstrates the dynamic nature of the organelle.
Secretory vesicle swelling is central to cell secretion, however the underlying mechanism of vesicle swelling, particularly synaptic vesicles, is not completely understood. The G αi3 -PLA2-mediated involvement of water channel AQP-1 in the regulation of secretory vesicle swelling in exocrine pancreas, and the G αo -mediated AQP-6 involvement in synaptic vesicle swelling in neurons, has previously been reported. Furthermore, the role of vH + -ATPase in neurotransmitter transport into synaptic vesicles, has also been shown. Using nanometer scale precision measurements of isolated synaptic vesicles, the present study reports for the first time, the involvement of vH + -ATPase in GTP-G αo -mediated synaptic vesicle swelling. Results from this study, demonstrate that the GTP-G αo -mediated vesicle swelling is vH + -ATPase-dependent, and pH sensitive. Zeta potential measurements of isolated synaptic vesicles further demonstrate, a bafilomycin-sensitive vesicle acidification, following the GTP-G αo -induced swelling stimulus. Since water channels are bidirectional, and the vH + -ATPase inhibitor bafilomycin decreases both the volume of isolated synaptic vesicles and GTP-mastoparan stimulated swelling, suggests vH + -ATPase to be upstream of AQP-6, in the pathway leading from G αo -stimulated swelling of synaptic vesicles. Vesicle acidification is therefore a prerequisite for AQP-6 mediated gating of water into synaptic vesicles.
To enable fusion between biological membranes, t-SNAREs and v-SNARE present in opposing bilayers, interact and assemble in a circular configuration forming ring-complexes, which establish continuity between the opposing membranes, in presence of calcium ions. The size of a t-/v-SNARE ring complex is dictated by the curvature of the opposing membrane. Hence smaller vesicles form small SNARE-ring complexes, as opposed to large vesicles. Neuronal communication depends on the fusion of 40–50 nm in diameter membrane-bound synaptic vesicles containing neurotransmitters at the nerve terminal. At the presynaptic membrane, 12–17 nm in diameter cup-shaped neuronal porosomes are present where synaptic vesicles transiently dock and fuse. Studies demonstrate the presence of SNAREs at the porosome base. Atomic force microscopy (AFM), electron microscopy (EM), and electron density measurement studies demonstrate that at the porosome base, where synaptic vesicles dock and transiently fuse, proteins, possibly comprised of t-SNAREs, are found assembled in a ring conformation. To further determine the structure and arrangement of the neuronal t-/v-SNARE complex, 50 nm t-and v-SNARE proteoliposomes were mixed, allowing t-SNARE-vesicles to interact with v-SNARE vesicles, followed by detergent solubilization and imaging of the resultant t-/v-SNARE complexes formed using both AFM and EM. Our results demonstrate formation of 6–7 nm membrane-directed self-assembled t-/v-SNARE ring complexes, similar to, but twice as large as the ring structures present at the base of neuronal porosomes. The smaller SNARE ring at the porosome base may reflect the 3–4 nm base diameter, where 40–50 nm in diameter v-SNARE-associated synaptic vesicle transiently dock and fuse to release neurotransmitters.
Approximately 11% smaller t-/v-SNARE ring complexes are generated using 50 nm cholesterolassociated vesicles, as opposed to vesicles containing L-α-Lysophosphatidylcholine (LPC), observed using atomic force microscopy (AFM). Circular dichroism (CD) spectroscopy demonstrates that as opposed to cholesterol, in presence of LPC, NSF-ATP induces disassembly of the β-sheet structures, but not the α-helical contents within the t-/v-SNARE complex.The chemistry of life processes is governed at the molecular level. For example, membranedirected self-assembly of a supramolecular ring complex is required for the establishment of continuity between opposing membrane compartments in cells 1,2 . Neurotransmission, and the secretion of hormones or digestive enzymes, all involve fusion of cellular membranes. At the nerve terminal, fusion involves conserved target membrane proteins SNAP-25 and syntaxin 1A, termed t-SNAREs, and synaptic vesicle-associated membrane protein VAMP2 or v-SNARE 3-5 . In the presence of Ca 2+ , when a v-SNARE-reconstituted liposome meets a t-SNARE-reconstituted vesicle, SNAREs in opposing membranes interact and self-assemble in a ring, establishing continuity between the compartments 1,2 . In the presence of ATP, this highly stable membrane-directed and self-assembled SNARE complex, can undergo disassembly in the presence of the soluble ATPase, N-ethylmaleimide-sensitive factor (NSF) 8 , 9. Cholesterol and lysophosphatidylcholine (LPC) are known to contribute to the negative and positive curvature of the cell membrane 6 , 7. Since cholesterol and LPC have been implicated in the promotion and inhibition of membrane fusion respectively10, their influence on membranedirected assembly and disassembly of the t-/v-SNARE ring complex was hypothesized. To test this hypothesis, structure of the t-/v-SNARE complex at nm resolution using atomic force microscopy (AFM), and at the molecular level, the secondary structure of SNAREs and their complex in membrane containing either cholesterol or LPC, was determined using circular dichroism (CD) spectroscopy9.Since vesicle size influences membrane curvature, a uniform vesicle population, prepared using a published 2 extrusion method, was used for the entire study. Two sets of 50 nm in diameter liposomes, one set containing cholesterol, and the other LPC, were reconstituted with either tSNAREs or v-SNARE for use (Figure 1). Surprisingly, examination at the nm level using AFM, demonstrated significant (p< 0.001) differences in SNARE ring size formed in the presence of cholesterol, compared to LPC. SNARE ring complexes formed using cholesterol-associated vesicles were found to be 6.89 nm, approximately 11% smaller than the 7.746 nm formed using LPC containing vesicles. Using CD spectroscopy, SNARE ring complexes formed either in the presence of cholesterol or LPC (Figure 2A,B) further demonstrate profound differences ( Table 1). As previously determined 9 , our results (Table 1) reveal, high α-helical content in t-SNARE and t-/v-SNARE complexes. However, in the pres...
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