The Drosophila dicistronic stoned locus encodes two distinctive presynaptic
proteins, Stoned A (STNA) and Stoned B (STNB); STNA is a novel protein wit
hout homology to known synaptic proteins, and STNB contains a domain with h
omology to the endocytotic protein AP50. Both Stoned proteins colocalize pr
ecisely with endocytotic proteins including the AP2 complex and Dynamin in
the "lattice network" characteristic of endocytotic domains in Drosophila p
resynaptic terminals. FM1-43 dye uptake studies in stoned mutants demonstra
te a striking decrease in the size of the endo-exo-cycling synaptic vesicle
pool and loss of spatial regulation of the vesicular recycling intermediat
es. Mutant synapses display a significant delay in vesicular membrane retri
eval after depolarization and neurotransmitter release. These studies sugge
st that the Stoned proteins play a role in mediating synaptic vesicle endoc
ytosis. We have documented previously a highly specific synaptic mislocaliz
ation and degradation of Synaptotagmin I in stoned mutants. Here we show th
at transgenic overexpression of Synaptotagmin I rescues stoned embryonic le
thality and restores endocytotic recycling to normal levels. Furthermore, o
verexpression of Synaptotagmin I in otherwise wild-type animals results in
increased synaptic dye uptake, indicating that Synaptotagmin I directly reg
ulates the endo-exo-cycling synaptic vesicle pool size. In parallel with re
cent biochemical studies, this genetic analysis strongly suggests that Ston
ed proteins regulate the AP2-Synaptotagmin I interaction during synaptic ve
sicle endocytosis. We conclude that Stoned proteins control synaptic transm
ission strength by mediating the retrieval of Synaptotagmin I from the plas
ma membrane.