Rab3-GEF Controls Active Zone Development at the Drosophila Neuromuscular Junction

Synaptic signaling involves the release of neurotransmitter from presynaptic active zones (AZs). Proteins that regulate vesicle exocytosis cluster at AZs, composing the cytomatrix at the active zone (CAZ). At the Drosophila neuromuscular junction (NMJ), the small GTPase Rab3 controls the distribution of CAZ proteins across release sites, thereby regulating the efficacy of individual AZs. Here we identify Rab3-GEF as a second protein that acts in conjunction with Rab3 to control AZ protein composition. At rab3-GEF mutant NMJs, Bruchpilot (Brp) and Ca(2+) channels are enriched at a subset of AZs, leaving the remaining sites devoid of key CAZ components in a manner that is indistinguishable from rab3 mutant NMJs. As the Drosophila homologue of mammalian DENN/MADD and Caenorhabditis elegans AEX-3, Rab3-GEF is a guanine nucleotide exchange factor (GEF) for Rab3 that stimulates GDP to GTP exchange. Mechanistic studies reveal that although Rab3 and Rab3-GEF act within the same mechanism to control AZ development, Rab3-GEF is involved in multiple roles. We show that Rab3-GEF is required for transport of Rab3. However, the synaptic phenotype in the rab3-GEF mutant cannot be fully explained by defective transport and loss of GEF activity. A transgenically expressed GTP-locked variant of Rab3 accumulates at the NMJ at wild-type levels and fully rescues the rab3 mutant but is unable to rescue the rab3-GEF mutant. Our results suggest that although Rab3-GEF acts upstream of Rab3 to control Rab3 localization and likely GTP-binding, it also acts downstream to regulate CAZ development, potentially as a Rab3 effector at the synapse.