Fig. 4: Modelling of CLIMP-63 palmitoylation dynamics.

a Simulation of the steady-state distribution of CLIMP-63 as predicted by the monomeric (M) model, with or without ZDHHC6 silencing. 0 and 1 superscripts indicate free or S-acylated CLIMP-63. b HA-CLIMP-63 and RFP-CLIMP-63 co-immunoprecipitation and 35S Cys/Met metabolic labelling of shCLIMP-63 HeLa cells expressing (WT or C100A mutant). Western blot analysis shows equivalent Co-IP of newly synthesised HA- and RFP-tagged CLIMP-63. c Western blot analysis of CLIMP-63 migrated on Blue Native or SDS-PAGE gels. d Blue Native gel analysis of shCLIMP-63 HeLa cells expressing CLIMP-63 luminal domain (LD) mutant with C- or N-terminal FLAG tag. e Western blot analysis of chromatography fractions of LD-CLIMP-63-FLAG on Blue Native or SDS-PAGE gels. Red square indicates fractions used for subsequent mass spectrometry analysis. f Intact mass LC-MS analysis under native-like conditions (Raw mass spectrum corresponding to LC peak between 2.7 and 3.2 min, magenta) of purified LD-CLIMP-63-FLAG. g, h Deconvolved mass spectra of the indicated LC peaks revealed: g a molecular mass between 173472 & 173786 Da, CLIMP-63 luminal trimers and h a mass of 57821 Da, monomers (green peak). i Schematic description of CLIMP-63 species and localization. E-Elementary trimer units, H-higher-order assemblies, and 0, 1 and 2 superscripts indicate zero, single, and double S-acylation of E within H. Acylation is catalysed by ZDHHC6 in the ER, and by ZDHHC2/5 at the plasma membrane. De-acylation is catalysed by APT2, both at the ER and at the plasma membrane. j Calibration and k validation of the model. The solid line represents the median of 100 simulated parameter sets; the shaded grey interval is defined by the 1st and the 3rd quartile; red points correspond to experimentally retrieved data points depicted in Fig. 3. l Simulation of the steady-state distribution of CLIMP-63 predicted by the oligomeric model (E + H), with and without ZDHHC6 silencing. m. Steady-state distribution of the different CLIMP-63 species as predicted for CLIMP-63 WT and C100A. n In silico (model) prediction of the total level of CLIMP-63 under control conditions (blue), ZDHHC6 silencing (grey), or ZDHHC6 overexpression (red). o Quantification of Western blot analysis of endogenous CLIMP-63 levels in control HeLa or ZDHHC6 KO cells overexpressing or not ZDHHC6. Values were normalised to the CLIMP-63 levels in control condition as 1. Results are mean ± SD and each data point corresponds to one biologically independent experiment—n > 17; p values were obtained by one-way ANOVA, Dunnet’s multiple comparison (***p = 0.0004, ****p < 0.0001). All simulation data sets represent the median, and error bars the first and third quartile or SD (bar charts) through the simulation of n = 100 models.