Fig. 6: Targeted depalmitoylation of specific protein complexes.
From: Engineered depalmitoylases enable selective manipulation of protein localization and function
a Schematic shows strategy for targeting FacePalm to individual protein complexes (FacePalm-it). FacePalmN is modified to include a nanobody that binds the desired target, here YFP. Activation of FacePalm-it results in selective depalmitoylation of YFP-tagged KChip2 but not Cer-tagged CaVβ2A. b Schematic shows the design of a constitutively active targeted depalmitoylases. Abhd17CT containing its catalytic domain is tethered to a nanobody targeting the desired target. c Confocal images of cells co-transfected with KChip2-YFP (green, left panels) and CaVβ2A-Cer (red pseudocolor, right panels). FacePalm-it has a YFP-selective nanobody targeting Kchip2. Scale bar, 10 µm. d Diary plot quantifies changes in cytosolic fluorescence of both KChip2-YFP (green dots and fit) and CaVβ2A-Cer (black dots and fit). Each dot, mean ± s.e.m from n = 23 cells from three transfections. e Confocal images of cells co-transfected with KChip2-YFP (green, left panels) and CaVβ2A-mcherry (red, right panels) along with FacePalm-it that targets a CaVβ2A-selective nanobody. Scale bar, 10 µm. f Diary plot shows changes in normalized cytosolic fluorescence for both KChip2-YFP (green dots and fit) and CaVβ2A-mcherry (black dots and fit). Each dot, mean ± s.e.m from n = 28 cells from three transfections. g At baseline, both Kchip2 and CaVβ2A are localized to the membrane. Scale bar, 10 µm. h Co-expression of Nb(YFP)-17ccat increases cytosolic localization of Kchip2, but not CaVβ2A. Scale bar, 10 µm. i Co-expression of Nb(CaVβ2A)-17ccat increases cytosolic localization of CaVβ2A but not Kchip2. Scale bar, 10 µm. j Cumulative distribution function of the ratio ζ = F̅cyt(CaVβ2A)/F̅cyt(Kchip2), where F̅cyt denotes the ratio of cytosolic fluorescence to total fluorescence in the cell. ζ > 1 denotes higher level of CaVβ2A in cytosol than Kchip2. Baseline (n = 70 cells); with Nb(YFP)-Abhd17Ccat (n = 166 cells); with Nb(CaVβ2A)-17ccat (n = 146 cells) from three independent transfections. ****p < 0.0001 by one-way ANOVA followed by Tukey’s multiple comparisons test. Source data are provided as a Source Data file.
