Abstract
Synaptic vesicles are storage organelles for neurotransmitters. They pass through a trafficking cycle and fuse with the pre-synaptic membrane when an action potential arrives at the nerve terminal. While molecular components and biophysical parameters of synaptic vesicles have been determined, our knowledge on the protein interactions in their membranes is limited. Here, we apply cross-linking mass spectrometry to study interactions of synaptic vesicle proteins in an unbiased approach without the need for specific antibodies or detergent-solubilisation. Our large-scale analysis delivers a protein network of vesicle sub-populations and functional assemblies including an active and an inactive conformation of the vesicular ATPase complex as well as non-conventional arrangements of the luminal loops of SV2A, Synaptophysin and structurally related proteins. Based on this network, we specifically target Synaptobrevin-2, which connects with many proteins, in different approaches. Our results allow distinction of interactions caused by ‘crowding’ in the vesicle membrane from stable interaction modules.
Highlights
Synaptic vesicles are storage organelles for neurotransmitters
From a technical point of view, chemical cross-linking uncovered, protein interactions in synaptic vesicles in an unbiased manner, i.e. we were able to identify protein interactions without the need for specific antibodies or detergent-based protein solubilisation. The latter is of particular importance, as detergent solubilisation yielded complexes differing in composition[29,30] suggesting that the protein complexes of synaptic vesicles are sensitive towards the detergent used
The assembly of ternary complexes or larger assemblies, has to be confirmed through other approaches, for instance western blotting of cross-linked complexes or pull-down experiments. This is true for interaction networks identified in synaptic vesicles as they pass through different functional states of the vesicle cycle and the assemblies fulfil the required functions at the various stages
Summary
Synaptic vesicles are storage organelles for neurotransmitters They pass through a trafficking cycle and fuse with the pre-synaptic membrane when an action potential arrives at the nerve terminal. Our large-scale analysis delivers a protein network of vesicle subpopulations and functional assemblies including an active and an inactive conformation of the vesicular ATPase complex as well as non-conventional arrangements of the luminal loops of SV2A, Synaptophysin and structurally related proteins. Based on this network, we target Synaptobrevin-2, which connects with many proteins, in different approaches. Additional abundant vesicle components are Synaptophysin, Synaptotagmin-1 and Synapsin-
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