At synapses, the release of neurotransmitter is regulated by molecular machinery that aggregates at specialized presynaptic release sites termed active zones. The complement of active zone proteins at each site is a determinant of release efficacy and can be remodeled to alter synapse function. The small GTPase Rab3 was previously identified as playing a novel role that controls the distribution of active zone proteins to individual release sites at the Drosophila neuromuscular junction. Rab3 has been extensively studied for its role in the synaptic vesicle cycle; however, the mechanism by which Rab3 controls active zone development remains unknown. To explore this mechanism, we conducted a mutational analysis to determine the molecular and structural requirements of Rab3 function at Drosophila synapses. We find that GTP-binding is required for Rab3 to traffick to synapses and distribute active zone components across release sites. Conversely, the hydrolytic activity of Rab3 is unnecessary for this function. Through a structure-function analysis we identify specific residues within the effector-binding switch regions that are required for Rab3 function and determine that membrane attachment is essential. Our findings suggest that Rab3 controls the distribution of active zone components via a vesicle docking mechanism that is consistent with standard Rab protein function.
CDR Region Mutation Brp Distribution Rescue
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CDR Region Mutation Rab3 Intensity
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deltaC Brp Distribution Rescue
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deltaC Rab3 Intensity
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N134I and T35N Brp Distribution Rescue
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N134I and T35N Rab3 Intensity
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N134I and T35N DN Phenotype
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Q80L Brp Distribution Rescue
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Q80L Rab3 Intensity
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Q80L Overexpression in WT
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Rab3-GAP Brp Area
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Switch Region Mutation Brp Distribution Rescue
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Switch Region Mutation Rab3 Intensity
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T53A Y83A and deltaC in WT
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Traces for mEJC Quantification
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Traces for EJC Quantification
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Traces for Facilitation Index Quantification
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