Fig. 3: An optogenetic actuator allows reversible manipulation of protein palmitoylation.
From: Engineered depalmitoylases enable selective manipulation of protein localization and function
a Schematic shows design of opto-depalm, an optogenetic actuator to manipulate protein palmitoylation. Abhd17NT is attached to eMagA while Abhd17CT is attached to eMagB. With blue light illumination, eMagA and eMagB dimerize resulting in reconstitution and activation of the Abhd17 holo-enzyme. In darkness, eMagA and eMagB dissociate rendering the enzyme inactive. b Confocal images show time-dependent changes in localization of CaVβ2A-mCherry in the absence (top row) or presence (bottom row) of opto-depalm. Blue light illumination from 0 to 60 min. Scalebar, 5 µm. c Diary plot quantifies normalized cytosolic fluorescence of CaVβ2A with (blue dots) opto-depalm or opto-depalm S/A (red dots). Black dots, CaVβ2A alone. Blue shaded region corresponds to period with blue light illumination. Each dot, mean ± s.e.m. n = 16 (no opto-depalm), n = 8 (with opto-depalm) and n = 13 (opto-depalm S/A mutant) cells from two independent transfections. d Acyl-RAC assay shows a reversible and time-dependent change in CaVβ2A palmitoylation upon photoactivation of opto-depalm. t = 0 min, prior to light-activation; t = 60 min, following light-activation; t = 180 min, recovery in darkness. HA hydroxylamine. Palmitoylated fraction and input are derived from the same experiment and processed in parallel. e Bar graph summary of relative band intensities from (d) confirms reversible depalmitoylation of CaVβ2A with opto-depalm. Each bar and error, mean ± s.e.m. from three independent trials, *p = 0.0286, one way ANOVA followed by Dunnett’s multiple comparison test. Source data are provided as a Source Data file.
