Abstract
The stoned locus of Drosophila melanogaster encodes two novel proteins, stonedA (STNA) and stonedB (STNB), both of which are expressed in the nervous system. Flies with defects at the stoned locus have abnormal behavior and altered synaptic transmission. Genetic interactions, in particular with the shibire (dynamin) mutation, indicated a presynaptic function for stoned and suggested an involvement in vesicle cycling. Immunological studies revealed colocalization of the stoned proteins at the neuromuscular junction with the integral synaptic vesicle protein synaptotagmin (SYT). We show here that stoned interacts genetically with synaptotagmin to produce a lethal phenotype. The STNB protein is found by co-immunoprecipitation to be associated with synaptic vesicles, and glutathione S-transferase pull-downs demonstrate an in vitro interaction between the micro2-homology domain of STNB and the C2B domain of the SYTI isoform. The STNA protein is also found in association with vesicles, and it too exhibits an in vitro association with SYTI. However, we find that the bulk of STNA is in a nonmembranous fraction. By using the shibire mutant to block endocytosis, STNB is shown to be present on some synaptic vesicles before exocytosis. However, STNB is not associated with all synaptic vesicles. We hypothesize that STNB specifies a subset of synaptic vesicles with a role in the synaptic vesicle cycle that is yet to be determined.