Fig. 4: Organelle-targeted depalmitoylation by FacePalm.
From: Engineered depalmitoylases enable selective manipulation of protein localization and function
a Schematic shows secretory pathway and strategy for targeting FacePalm to specific organelles. FacePalmN is modified to contain targeting sequences to distinct organelles. Upon addition of rapamycin, FacePalmC containing the catalytic domain is recruited to the desired subcellular location, resulting in increased depalmitoylation of proteins from these regions. Targeting sequences for each subcellular location are noted in italics. Activation of ER-targeted FacePalm results in minimal change in localization of CaVβ2A-YFP. Confocal images (b). Scale bar, 10 µm. Diary plot shows normalized cytosolic fluorescence (c). Each dot and error, mean ± s.e.m from n = 24 cells from three independent transfections. d, e Activation of Golgi-targeted FacePalm results in a modest increase in cytosolic fluorescence. Format as in (b, c). Each dot and error, mean ± s.e.m, n = 25 cells from three independent transfections. Scale bar, 10 µm. f, g Activation of plasma-membrane localized FacePalm results in a strong increase in cytosolic fluoresce of CaVβ2A-YFP. Format as in (b, c). Each dot and error, mean ± s.e.m, n = 22 cells from three independent transfections. Scale bar, 10 µm. h, i Activation of early-endosome (Rab5) localized FacePalm also evokes a strong increase in cytosolic fluoresce of CaVβ2A-YFP. Format as in (b, c). Each dot and error, mean ± s.e.m, n = 17 cells from three independent transfections. Scale bar, 10 µm. j, k Activation of late-endosome localized FacePalm evoked a modest increase in cytosolic fluorescence. Format as in (b, c). Each dot and error, mean ± s.e.m, n = 20 cells from three independent transfections. Scale bar, 10 µm. l Bar graph summarizes maximal change in cytosolic fluorescence in response to activation of FacePalm localized to distinct domains. Each bar, mean ± s.e.m. *p < 0.05 (ER vs Golgi p = 0.0199, Rab5 vs Rab7 p = 0.0275), *** p = 0.0009 (Golgi vs PM), and **** p < 0.0001 (ER vs PM) by one way ANOVA followed by Dunnett’s T3 multiple comparison’s test. Source data are provided as a Source Data file.
