Botulinum neurotoxins (BoNTs) block neurotransmitter release through their specific proteolysis of the proteins responsible for vesicle exocytosis. Paradoxically, two serotypes of BoNTs, A and E, cleave the same molecule, synaptosome-associated protein with relative molecular mass 25K , and yet they cause synaptic blockade with very different properties. Here we compared the action of BoNTs A and E on the plasma membrane fusion machinery composed of syntaxin and SNAP-25. We now show that the BoNT/A-cleaved SNAP-25 maintains its association with two syntaxin isoforms in vitro, which is mirrored by retention of SNAP-25 on the plasma membrane in vivo. In contrast, BoNT/E severely compromises the ability of SNAP-25 to bind the plasma membrane syntaxin isoforms, leading to dissociation of SNAP-25. The distinct properties of botulinum intoxication, therefore, can result from the ability of shortened SNAP-25 to maintain its association with syntaxins-in the case of BoNT/A poisoning resulting in unproductive syntaxin/SNAP-25 complexes that impede vesicle exocytosis.
SNAP-25 (25 kDa synaptosome-associated protein) is found in cells that release neurotransmitters and hormones, and plays a central role in the fusion of secretory vesicles with the plasma membrane. SNAP-25 has been shown to interact specifically with syntaxin 1, a 35 kDa membrane protein, to mediate the fusion process. Here, we investigated whether other known syntaxin isoforms found at the plasma membrane can serve as binding partners for SNAP-25 in vivo. In our analysis, we employed rat phaeochromocytoma PC12 cells that are often used as a model of neuronal functions. We now show that these cells contain large amounts of SNAP-25, which interacts not only with syntaxin 1, but also with ubiquitous syntaxins 2, 3 and 4. The plasma membrane syntaxins appear to occupy complementary domains at the plasma membrane. In defined reactions, the ubiquitous plasma membrane syntaxin isoforms, when in binary complexes with SNAP-25, readily bound vesicular synaptobrevin to form SDS-resistant SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) complexes implicated in membrane fusion. However, vesicular synaptotagmin and cytosolic complexin, both implicated in the fusion process, exhibited differential ability to interact with the SNARE complexes formed by syntaxins 1-4, suggesting that the plasma membrane syntaxins may mediate vesicle fusion events with different properties.
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