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Synaptic vesicles are constitutively active fusion machines that function independently of Ca2+.

ABSTRACT: In neurons, release of neurotransmitter occurs through the fusion of synaptic vesicles with the plasma membrane. Many proteins required for this process have been identified, with the SNAREs syntaxin 1, SNAP-25, and synaptobrevin thought to constitute the core fusion machinery. However, there is still a large gap between our understanding of individual protein-protein interactions and the functions of these proteins revealed by perturbations in intact synaptic preparations. To bridge this gap, we have used purified synaptic vesicles, together with artificial membranes containing core-constituted SNAREs as reaction partners, in fusion assays.By using complementary experimental approaches, we show that synaptic vesicles fuse constitutively, and with high efficiency, with proteoliposomes containing the plasma membrane proteins syntaxin 1 and SNAP-25. Fusion is inhibited by clostridial neurotoxins and involves the formation of SNARE complexes. Despite the presence of endogenous synaptotagmin, Ca(2+) does not enhance fusion, even if phosphatidylinositol 4,5-bisphosphate is present in the liposome membrane. Rather, fusion kinetics are dominated by the availability of free syntaxin 1/SNAP-25 acceptor sites for synaptobrevin.Synaptic vesicles are constitutively active fusion machines, needing only synaptobrevin for activity. Apparently, the final step in fusion does not involve the regulatory activities of other vesicle constituents, although these may be involved in regulating earlier processes. This is particularly relevant for the calcium-dependent regulation of exocytosis, which, in addition to synaptotagmin, requires other factors not present in the vesicle membrane. The in vitro system described here provides an ideal starting point for unraveling of the molecular details of such regulatory events.

SUBMITTER: Holt M

PROVIDER: S-EPMC2481520 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

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Synaptic vesicles are constitutively active fusion machines that function independently of Ca2+.

Holt Matthew M Riedel Dietmar D Stein Alexander A Schuette Christina C Jahn Reinhard R

Current biology : CB 20080501 10

<h4>Background</h4>In neurons, release of neurotransmitter occurs through the fusion of synaptic vesicles with the plasma membrane. Many proteins required for this process have been identified, with the SNAREs syntaxin 1, SNAP-25, and synaptobrevin thought to constitute the core fusion machinery. However, there is still a large gap between our understanding of individual protein-protein interactions and the functions of these proteins revealed by perturbations in intact synaptic preparations. To ...[more]

PMID: 18485705

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Project description:The CCR5 chemokine receptor is a rhodopsin-like G protein-coupled receptor that mediates the effects of pro-inflammatory β-chemokines. CCR5 is also the major co-receptor for entry of human immunodeficiency virus (HIV) into human cells. G protein-coupled receptors exist in ensembles of active and inactive conformations. Active receptor conformations can be stabilized by mutations. Although binding of the HIV envelope protein to CCR5 stimulates cellular signaling, the CCR5 conformation that induces fusion of the viral membrane with cellular membranes is not known. We mutated conserved amino acids to generate constitutively active CCR5 receptors, which are stabilized in active conformations, and tested the ability of constitutively active CCR5 receptors to mediate HIV envelope-directed membrane fusion. Mutation of the Asp³·⁴⁹(¹²⁵) and Arg⁶·³²(²²⁵) residues of CCR5 did not cause constitutive activity, but Lys or Pro substitutions for Thr²·⁵⁶(⁸²), in the TxP motif, caused high basal inositol phosphate signaling. Signaling did not increase in response to MIP-1β, suggesting that the Thr²·⁵⁶(⁸²) mutants were fully stabilized in active conformations. The Thr²·⁵⁶(⁸²)Lys mutation severely decreased cell surface CCR5 expression. Combining the Thr²·⁵⁶(⁸²)Lys mutation with an Arg⁶·³²(²²⁵)Gln mutation partially reversed the decrease in expression. Mutants with Thr²·⁵⁶(⁸²)Lys substitutions were poor mediators of HIV envelope-directed membrane fusion, but mutants with the Thr²·⁶⁵(⁸²)Pro substitution exhibited full co-receptor function. Our results suggest that the Thr²·⁶⁵(⁸²)Lys and Thr²·⁶⁵(⁸²)Pro mutations stabilize distinct constitutively active CCR5 conformations. Lys in position 2.65(82) stabilizes activated receptor conformations that appear to be constitutively internalized and do not induce envelope-dependent membrane fusion, whereas Pro stabilizes activated conformations that are not constitutively internalized and fully mediate envelope-directed membrane fusion.

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Synaptic vesicles are constitutively active fusion machines that function independently of Ca2+.

Publications

Synaptic vesicles are constitutively active fusion machines that function independently of Ca2+.

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