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A v-SNARE participates in synaptic vesicle formation mediated by the AP3 adaptor complex

Abstract

Reconstitution of synaptic vesicle formation in vitro has revealed a pathway of synaptic vesicle biogenesis from endosomes that requires the heterotetrameric adaptor complex AP3. Because synaptic vesicles have a distinct protein composition, the AP3 complex should selectively recognize some or all of the synaptic vesicle proteins. Here we show that one element of this recognition process is the v-SNARE, VAMP-2, because tetanus toxin, which cleaves VAMP-2, inhibited the formation of synaptic vesicles and their coating with AP3 in vitro. Mutant tetanus toxin and botulinum toxins, which cleave t-SNAREs, did not inhibit synaptic vesicle production. AP3-containing complexes isolated from coated vesicles could be immunoprecipitated by a VAMP-2 antibody. These data imply that AP3 recognizes a component of the fusion machinery, which may prevent the production of inert synaptic vesicles.

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Figure 1: Inhibition of VAMP-2 targeting into synaptic vesicles by tetanus toxin in a cell-free assay.
Figure 2: Inhibition of synaptic vesicle biogenesis from donor membranes treated with tetanus toxin using two different assays.
Figure 3: The t-SNAREs syntaxin 1a and SNAP-25 are not involved in synaptic vesicle biogenesis.
Figure 4: Recruitment of AP3 by VAMP-2.

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Acknowledgements

We thank Reinhard Jahn for the gift of VAMP-2 (69.1) and synaptogyrin (R21) antibodies, Dr. H. Niemann for the tetanus and botulinum toxin cDNAs, Dr. C. Barnstable for the syntaxin 1a antibody (HPC-1), Dr. D. Shields for wild-type human ARF1 cDNA and Dr. J.S. Bonifacino for the ß-NAP GST-fusion protein. This research was supported by grants to R.B.K. from the National Institutes of Health, to N.S. from the Boyer fellowship and to V.F. from the Fogarty fellowship.

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Correspondence to Regis B. Kelly.

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Salem, N., Faúndez, V., Horng, JT. et al. A v-SNARE participates in synaptic vesicle formation mediated by the AP3 adaptor complex. Nat Neurosci 1, 551–556 (1998). https://doi.org/10.1038/2787

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