Overlapping functions of stonin 2 and SV2 in sorting of the calcium sensor synaptotagmin 1 to synaptic vesicles

Neurotransmission involves the calcium-regulated exocytic fusion of synaptic vesicles (SVs) and the subsequent retrieval of SV membranes followed by reformation of properly sized and shaped SVs. An unresolved question is whether each SV protein is sorted by its own dedicated adaptor or whether sorting is facilitated by association between different SV proteins. We demonstrate that endocytic sorting of the calcium sensor synaptotagmin 1 (Syt1) is mediated by the overlapping activities of the Syt1-associated SV glycoprotein SV2A/B and the endocytic Syt1-adaptor stonin 2 (Stn2). Deletion or knockdown of either SV2A/B or Stn2 results in partial Syt1 loss and missorting of Syt1 to the neuronal surface, whereas deletion of both SV2A/B and Stn2 dramatically exacerbates this phenotype. Selective missorting and degradation of Syt1 in the absence of SV2A/B and Stn2 impairs the efficacy of neurotransmission at hippocampal synapses. These results indicate that endocytic sorting of Syt1 to SVs is mediated by the overlapping activities of SV2A/B and Stn2 and favor a model according to which SV protein sorting is guarded by both cargo-specific mechanisms as well as association between SV proteins.Neurotransmission is based on the calcium-triggered fusion of neurotransmitter-filled synaptic vesicles (SVs) with the presynaptic plasma membrane. To sustain neurotransmitter release, neurons have evolved mechanisms to retrieve SV membranes and to reform SVs locally within presynaptic nerve terminals. How SVs are reformed and maintain their compositional identity (1, 2) is controversial (3–5). One possibility is that upon fusion SV proteins remain clustered at the active zone—that is, by association between SV proteins—and are retrieved via “kiss-and-run” or ultrafast endocytosis (6), thereby alleviating the need for specific sorting of individual SV proteins. Alternatively, if SVs lose their identity during multiple rounds of exo-/endocytosis (7, 8), specific mechanisms exist to orchestrate high-fidelity SV protein sorting, either directly at the plasma membrane via slow clathrin-mediated endocytosis (CME) or at endosome-like vacuoles generated by fast clathrin-independent membrane retrieval (5, 9). Endocytic adaptors for SV protein sorting include the heterotetrameric adaptor protein complex 2 (AP-2) (9), the synaptobrevin 2/VAMP2 adaptor AP180 (10), and the AP-2μ–related protein stonin 2 (Stn2), a specific sorting adaptor for the SV calcium sensor synaptotagmin 1 (Syt1) (8, 11). Although genetic inactivation of the Stn2 orthologs Stoned B and Unc41 in flies and worms is lethal due to defective neurotransmission caused by degradation and missorting of Syt1 (12, 13), Stn2 knockout (KO) mice are viable and able to internalize Syt1, albeit with reduced fidelity of sorting (14). Thus, mammalian synapses, in contrast to invertebrates, have evolved mechanisms to sort Syt1 in the absence of its specific sorting adaptor Stn2. One possibility is that Syt1 sorting in addition to its direct recognition by Stn2 is facilitated by complex formation with other SV proteins. Likely candidates for such a piggyback mechanism are the SV2 family of transmembrane SV glycoproteins (15, 16), which might regulate Syt1 function either via direct interaction (17, 18) or by facilitating its binding to AP-2 (19). Apart from the distantly related SVOP protein (20), no close SV2 homologs exist in invertebrates, suggesting that SV2 fulfills a unique function at mammalian synapses. KO of SV2A or combined loss of its major A and B isoforms in mice causes early postnatal lethality due to epileptic seizures (21, 22), impaired neurotransmission (23, 24), and defects in Syt1 trafficking (25), whereas SV2B KO mice are phenotypically normal (17). Given that SV2A in addition to its association with Syt1 binds to endocytic proteins including AP-2 and Eps15 (25), SV2 would be a likely candidate for mediating Syt1 sorting to SVs.Here we demonstrate that endocytic sorting of Syt1 is mediated by the overlapping activities of SV2A/B and Stn2. Deletion or knockdown of either SV2A/B or Stn2 results in partial Syt1 loss and missorting of Syt1 to the neuronal surface, whereas deletion of both SV2A/B and Stn2 dramatically exacerbates this phenotype, resulting in severely impaired basal neurotransmission. Our results favor a model according to which SV protein sorting is guarded by both cargo-specific mechanisms as well as association between SV proteins.