Rolling blackout, a newly identified PIP₂-DAG pathway lipase required for Drosophila phototransduction

Supplementary Fig. 1 (pdf 267K)
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.

Supplementary Fig. 2 (pdf 301K)
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).

Supplementary Fig. 3 (pdf 41K)
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 rbo^ts1 is indicated.

Supplementary Fig. 4 (pdf 231K)
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), rbo^ts1/rbo² mutants (rbo) and PLC norpA null mutants (norpA^P24) 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 norpA^P24 had negligible PLC activity, <1% of either WT or rbo mutants.

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