Abstract
The rolling blackout (rbo) gene encodes an integral plasma membrane lipase required for Drosophila phototransduction. Photoreceptors are enriched for the RBO protein, and temperature-sensitive rbo mutants show reversible elimination of phototransduction within minutes, demonstrating an acute requirement for the protein. The block is activity dependent, indicating that the action of RBO is use dependent. Conditional rbo mutants show activity-dependent depletion of diacylglycerol and concomitant accumulation of phosphatidylinositol phosphate and phosphatidylinositol 4,5-bisphosphate within minutes of induction, suggesting rapid downregulation of phospholipase C (PLC) activity. The RBO requirement identifies an essential regulatory step in G-protein-coupled, PLC-dependent inositol lipid signaling mediating activation of TRP and TRPL channels during phototransduction.
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Acknowledgements
We thank the Drosophila Bloomington Stock Center for supplying stocks; T. Jongens for cmp44E RFII pBluescript vector; T. Jongens, S. Chandrashekaran, C. Montell, H. Li, R. Hardie and W. Pak for flies, including cmp44E, stmA, trp343, trpl302 and norpA mutants; C. Sanders for DAG kinase; R. Shortridge for advice about PLC assays; R. Mohrmann for discussions, useful comments and help in generating figures and L. Yang for assistance in sequencing mutant alleles. This study was supported by US National Institutes of Health grant GM54544 to K.B.
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Supplementary information
Supplementary Fig. 1
stmA, cmp44E and rbo mutants are allelic to each other, based on both lethality and conditional blindness complementation studies. (a)"*" indicates phenotype was fully rescued by genomic rbo-egfp expression. A single naming strategy was adopted for all mutant alleles of the locus, as indicated. (b) Representative traces showing the loss (top) and genomic rescue (bottom) of phototransduction in rbo mutants. (PDF 266 kb)
Supplementary Fig. 2
RBO dendrogram. A dendrogram generated by Clustal W representing the relationship of RBO homologs from multiple species. A single rbo gene is present in both Drosophila melanogaster (Dmrbo) and Anapheles gambiae (Agrbo). The closest homologs are two genes present in both human (Hsrbo1,2) and mouse (Mmrbo1,2), and, more distantly, a single gene in C. elegans (Cerbo). Homologs are present in yeast (Scrbo). The outlier groups contain homologs from Arabidopsis (Atrbo) and rice (Osrbo). (PDF 300 kb)
Supplementary Fig. 3
RBO is a predicted lipase. The RBO protein contains the domain structure of a lipolytic enzyme. Three highly-conserved lipase functional motifs (His, Ser and Asp/Glu active sites) contain the catalytic triad of His, Ser, and Asp/Glu residues, respectively. The consensus motif "GXSXG" is found in the Ser active site. The mutation site in rbots1 is indicated. (PDF 40 kb)
Supplementary Fig. 4
PLC activity is normal in rbo mutant head extracts. (a) Schematic diagram of the Drosophila phototransduction pathway. Light activation of rhodopsin (Rh) activates Gq, which activates PLC. PLC hydrolyzes PIP2 into DAG and IP3 in the microvillar membrane. DAG either directly activates TRP/TRPL channels or is further hydrolyzed by DAG lipase to produce PUFAs, which in turn activate TRP/TRPL channels. DAG Kinase (DGK; I) converts DAG to phosphatidic acid (PA), which is then recycled to synthesize CDP-DAG via CDP-DAG synthase (II) in the subrhabdomeric cisternae. CDP-DAG is converted to PI by phosphatidylinositol synthase (III) to form PI. PI is then transported back to the microvillar membrane by PI transport protein (IV) and serially phosphorylated by PI kinase and PIP kinase (V,VI) to reproduce PIP2. Dotted lines indicate hypothetical pathways. (b) PLC activity assay from head extracts (see Methods) of wildtype (WT), rbots1/rbo2 mutants (rbo) and PLC norpA null mutants (norpAP24) at 25°C and 37°C, as indicated. Higher temperature caused a consistent increase in PLC activity levels in both WT and rbo mutants. The two genotypes were indistinguishable. The PLC null norpAP24 had negligible PLC activity, <1% of either WT or rbo mutants. (PDF 230 kb)
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Huang, FD., Matthies, H., Speese, S. et al. Rolling blackout, a newly identified PIP2-DAG pathway lipase required for Drosophila phototransduction. Nat Neurosci 7, 1070–1078 (2004). https://doi.org/10.1038/nn1313
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DOI: https://doi.org/10.1038/nn1313
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