Cellular Role Of The Drosophila EFR3, Rolling Blackout (RBO) In Synaptic Transmission.

Abstract

Rolling Blackout (RBO), a Drosophila EFR3 homolog, is an integral membrane protein predicted to be a lipase. A conditional temperature-sensitive (TS) mutant (rbots) displays paralysis following a 25-37oC temperature shift, an impairment previously attributed to blocked synaptic vesicle exocytosis. rbots displays a synergistic interaction with a dominant positive Syntaxin-1A TS allele, syx3-69. At neuromuscular synapses, rbots shows a defect in styryl FM dye endocytosis, and rbots;syx3-69 double mutants display a synergistic, more severe endocytosis impairment. rbots synapses in primary brain culture show defective FM endocytosis. Nephrocyte Garland cells show the same endocytosis defect in tracer uptake assays. Ultrastructurally, rbots displays a defect in tracer uptake into endosomes in both neuronal and non-neuronal cells. At the rbots synapse, there is total blockade of endosome formation possibly via activity-dependent bulk endocytosis. A point mutation in the putative lipase active site fails to rescue embryonic lethality of the null mutants and garland cell endocytosis defects in the TS background, suggesting that the mutation disrupts some vital aspect of protein function and that lipase activity may be required for membrane trafficking and viability. However, the putative lipase-dead mutant is able to fully rescue paralysis and NMJ endocytic defects in rbots possibly indicative of interallelic complementation. Further, we identified a synthetic lethal interaction between rbots and the dynamin GTPase mutant shits1. In all assays, both in non-neuronal cells and neuronal synapses, shits1; rbots phenocopies shits1 endocytic defects, indicating that dynamin and RBO act in the same pathway. Together, these results demonstrate that RBO is required for dynamin-dependent bulk endocytosis/macropinocytosis in both neuronal and non-neuronal cells, and at the synapse this mechanism is responsive to the rate of Syntaxin-1A-dependent exocytosis.