Drosophila stoned proteins regulate the rate and fidelity of synaptic vesicle internalization

At an initial step during synaptic vesicle recycling, dynamin and adaptor proteins mediate the endocytosis of synaptic vesicle components from the plasma membrane. StonedA and stonedB, novel synaptic proteins encoded by a single Drosophila gene, have predicted structural similarities to adaptors and other proteins implicated in endocytosis. Here, we test possible roles of the stoned proteins in synaptic vesicle internalization via analyses of third instar larval neuromuscular synapses in two Drosophila stoned (stn) mutants, stn(ts) and stn(8P1). Both mutations reduce presynaptic levels of stonedA and stonedB, although stn ts has relatively weak effects. The mutations cause retention of synaptic vesicle proteins on the presynaptic plasma membrane but do not alter the levels or distribution of endocytosis proteins, dynamin, alpha -adaptin, and clathrin. In addition, stn(8P1) mutants exhibit depletion and enlargement of synaptic vesicles. To determine whether these defects arise from altered synaptic vesicle endocytosis or from defects in synaptic vesicle biogenesis, we implemented new methods to assess directly the efficiency of synaptic vesicle recycling and membrane internalization at Drosophila nerve terminals. Behavioral and electrophysiological analyses indicate that stn(ts), an allele with normal evoked release and synaptic vesicle number, enhances defects in synaptic vesicle recycling shown by Drosophila shi(ts) mutants. A dye uptake assay demonstrates that slow synaptic vesicle recycling in stn(ts) is accompanied by a reduced rate of synaptic vesicle internalization after exocytosis. These observations are consistent with a model in which stonedA and stonedB act to facilitate the internalization of synaptic vesicle components from the plasma membrane.