Rolling blackout (RBO) is a Drosophila EFR3 integral membrane lipase. A conditional temperature-sensitive (TS) mutant (rbots) displays paralysis within minutes following a temperature shift from 25°C to 37°C, an impairment previously attributed solely to blocked synaptic-vesicle exocytosis. However, we found that rbots displays a strong synergistic interaction with the Syntaxin-1A TS allele syx3-69, recently shown to be a dominant positive mutant that increases Syntaxin-1A function. At neuromuscular synapses, rbots showed a strong defect in styryl-FM-dye (FM) endocytosis, and rbots;syx3-69 double mutants displayed a synergistic, more severe, endocytosis impairment. Similarly, central rbots synapses in primary brain culture showed severely defective FM endocytosis. Non-neuronal nephrocyte Garland cells showed the same endocytosis defect in tracer-uptake assays. Ultrastructurally, rbots displayed a specific defect in tracer uptake into endosomes in both neuronal and non-neuronal cells. At the rbots synapse, there was a total blockade of endosome formation via activity-dependent bulk endocytosis. Clathrin-mediated endocytosis was not affected; indeed, there was a significant increase in direct vesicle formation. Together, these results demonstrate that RBO is required for constitutive and/or bulk endocytosis and/or macropinocytosis in both neuronal and non-neuronal cells, and that, at the synapse, this mechanism is responsive to the rate of Syntaxin-1A-dependent exocytosis.

Phosphatidylethanolamine (PtdEtn) is one of the most abundant phospholipids in many animal cell types. The Drosophila easily shocked (eas2) mutant, used as an epilepsy model, is null for the PtdEtn biosynthetic enzyme, ethanolamine kinase. This mutant displays bang sensitive paralysis, and was previously shown to have decreased levels of PtdEtn. We have developed a highly selective and sensitive measurement strategy using ion mobility-mass spectrometry for the relative quantitation of intact phospholipid species directly from isolated brain tissue of eas mutants. Over 1200 distinct lipid signals are observed and within this population 38, including PtdEtn, phosphatidylinositol (PtdIns) and phosphatidylcholine (PtdCho) species are identified to have changed significantly (p<0.03) between mutant and control tissue. This method has revealed for the first time the structural complexity and interconnectedness of specific PtdEtn and PtdIns lipid species within tissue, and provides great molecular detail compared to traditionally used detection techniques.

Forward genetic screens have identified numerous proteins with critical roles in neurotransmission. One particularly fruitful screening target in Drosophila has been TS (temperature-sensitive) paralytic mutants, which have revealed proteins acutely required in neuronal signalling. In the present paper, we review recent insights and current questions from one recently cloned TS paralytic mutant, rbo (rolling blackout). The rbo mutant identifies a putative integral lipase of the pre-synaptic plasma membrane that is required for the SV (synaptic vesicle) cycle. Identification of this mutant adds to a growing body of evidence that lipid-modifying enzymes locally control specialized lipid microenvironments and lipid signalling pathways with key functions regulating neurotransmission strength. The RBO protein is absolutely required for phospholipase C signalling in phototransduction. We posit that RBO might be required to regulate the availability of fusogenic lipids such as phosphatidylinositol 4,5-bisphosphate and diacylglycerol that may directly modify membrane properties and/or activate lipid-binding fusogenic proteins mediating SV exocytosis.

There was an error published in J. Cell Sci. 122, 114-125.In the left-hand panel of Fig. 7C (left), the control condition at 37°C was mistakenly shown with an example at 25°C. This error is corrected below with a new Fig. 7C (left) showing the control 37°C condition. The figure legend, results and interpretation for this work have not been altered in any way.The corresponding author apologizes for this error.

Drosophila temperature-sensitive rolling blackout (rbo ts ) mutants display a total block of endocytosis in nonneuronal cells and a weaker, partial defect at neuronal synapses. RBO is an integral plasma membrane protein and is predicted to be a serine esterase. To determine if lipase activity is required for RBO function, we mutated the catalytic serine 358 to alanine in the G-X-S-X-G active site, and assayed genomic rescue of rbo mutant non-neuronal and neuronal phenotypes. The lipid composition of the plasma membrane determines its flexibility, curvature and trafficking properties. Membrane retrieval endocytosis mechanisms are critically dependent on the temporally regulated focal accumulation of specific lipids. Numerous endocytic proteins have selective domains that recognize different membrane lipids. Pleckstrin homology (PH) and AP180 N-terminal homology/epsin N-terminal homology (ANTH/ENTH) domains bind acidic phospholipids such as phosphatidylinositol (4,5)-bisphosphate (PIP 2 ). PIP 2 is required for several steps in endocytic vesicle retrieval including clathrin coat nucleation, vesicle budding, scission and uncoating (1,2). The recruitment of endocytic adaptor proteins AP2 and AP180 requires PIP 2 . Bar (Bin Amphiphysin RVS) domain containing proteins such as endophilin and amphiphysin also binds negatively charged membrane lipids and facilitates membrane curvature during vesicle formation (3,4). The GTPase dynamin PH domain also binds PIP 2 (5), and PH domain mutations inhibit dynamin function to block vesicle endocytosis (6,7). Mutations that decrease brain PIP 2 levels result in delayed endocytosis and slower vesicle recycling kinetics (2).We previously reported the cloning and characterization of the Drosophila gene rolling blackout (rbo), also known as conserved membrane protein at 44E (cmp44E) and stambhA (stmA) (8,9). Null rbo mutants are 100% late embryonic lethal with zero escapers, whereas a conditional temperature-sensitive (TS) mutant (rbo ts ) paralyzes within minutes at restrictive temperature and acutely blocks synaptic transmission. Conditional rbo ts mutants display an activity-dependent complete loss of phospholipase C (PLC)-mediated phototransduction and consistently, at the molecular level, rapidly accumulate phosphatidylinositol-4-phosphate (PI4P) and PIP 2 , and concomitantly lose diacylglycerol (DAG), within minutes after exposure to restrictive temperature (8). RBO is an integral transmembrane plasma membrane protein with significant domain homology to membrane lipases, containing the pentapeptide motif G-X-S-X-G in which the central serine (358) functions as the catalytic active site together with a histidine (H289) and an aspartate (D719), forming a serine esterase catalytic triad similar to sn-1 DAG lipase and neuropathy target esterase/swiss cheese (10,11). These data strongly suggest that RBO functions directly as a lipase in the PIP 2 -DAG pathway. On the other hand, the yeast RBO homolog EFR3 (Pho eighty five requiring 3) is proposed to function as a scaffolding protein...