Extended Data Fig. 6: Mutations identified in various superPLD clones.
From: Activity-based directed evolution of a membrane editor in mammalian cells
a, PLD mutant clones are shown in order of PLD activity in cells determined by IMPACT (increasing from left to right), and black dots indicate the presence of a particular point mutation in that PLD mutant. Mutants with identical sets of mutations are not shown in the plot. The mutated residues are coloured based on conservation across PLDs from different species, determined by ConSurf;1,2,3 red: high conservation, white: medium, blue: low. b, Activity assay of PLD mutants containing different combinations of four commonly occurring mutations that were generated in the PLDWT background (A258T, G381V, G429D and T450A). Cells expressing plasma membrane-targeted optoPLD with the indicated set of mutations were labelled with IMPACT using 1 mM azidopropanol and 1 µM BCN-BODIPY. IMPACT fluorescence intensity normalized to optoPLD expression was determined by flow cytometry, and the relative values for each mutant compared to the PLDWT are plotted as relative IMPACT labelling (n = 2 independent transfection replicates). The effect of each mutation occurred mostly in a combinatorial manner (that is, most mutations exhibited multiplicative effects in either increasing (G381V, T450A) or slightly decreasing (A258T) the activity, though G429D slightly increased activity alone but had negligible effects when combined with other mutations).
