Extended Data Fig. 1: Drosophila dOCRL displays a PI(4,5)P2 phosphatase activity in vitro.
From: Inducible depletion of PI(4,5)P2 by the synthetic iDePP system in Arabidopsis
a, Alignment of (i) Homo sapiens OCRL (HsOCRL or OCRL1) truncation (HsOCRL234-539) successfully used in optogenetic systems in mammalian cells, and (ii) of Drosophila melanogaster dOCRL. We were concerned that this domain, that underwent natural selection in an endotherm organism with set-point temperature of 37 °C, would be less active in Arabidopsis thaliana, a plant of temperate climate which we usually grow at 21 °C. Therefore, we decided to use the phosphatase domain of dOCRL, Drosophila melanogaster homolog, as Drosophila are ectotherm and live at ~ 20 °C. Alignment of OCRL1234-539 and dOCRL allowed us to identify dOCRL168-509 as the homolog region of OCRL1 generally used in optogenetic systems. b, Coomassie blue staining monitoring purification of recombinant dOCRL phosphatase domain (His-SUMO-dOCRL168-509-His) from Escherichia coli. We first assessed dOCRL activity, which has not been reported so far, even though in vivo data suggest a conserved PI(4,5)P2 5-phosphatase activity. We ordered a synthetic gene corresponding to dOCRL168-509 and codon optimized for expression in Arabidopsis. We cloned a recombinant dOCRL protein (His-SUMO-dOCRL168-509-His) tagged with two 6-histidine tags and a SUMO tag, making it suitable for expression in Escherichia coli and purification. We purified His-SUMO-dOCRL168-509-His using a cobalt resin-based affinity chromatography and protein concentration IN: input, lysate of bacteria induced for the expression of His-SUMO- dOCRL168-509-His; FL: flow through; W: wash; E1 to E3: elution fraction 1 to 3; C1 and C2: concentrated fractions from pulled E1, E2 and E3. C1 and C2 were obtained separately on different days. His-SUMO-dOCRL168-509-His expected molecular weight is 54.8 kD; 3 Replicates (c) Malachite green phosphatase assay on His-SUMO-dOCRL169-509-His using short chain water-soluble phosphoinositides. We subjected the purified protein fractions C1 and C2 to Malachite Green Phosphatase assays, that detects phosphate released by dephosphorylations, in presence of short chain (diC:8) water-soluble phosphoinositides. Note that we only assessed dOCRL168-509 activity toward phosphoinositides reported in plant cells. Replicate 1 correspond to C1 fraction. Replicate 2 to C2 fractions. Mock: no phosphoinositide added.
