Figure 1. ORAI1 is S-acylated at cysteine C143.

(A) ORAI1 immunoblot of HeLa cells treated with PEG-5k to label S-acylation sites after exposure to NEM to block free thiols and then to hydroxylamine (HA) to break acyl-thioester bonds. (B) Western blot and corresponding autoradiogram of HeLa cells labelled for 2 hr with ³H-palmitic acid with or without HA and immunoprecipitated with anti-ORAI1. (C, D) Western blots and corresponding autoradiograms of HeLa (C) and RPE-1 (D) cells expressing the indicated YFP-tagged ORAI1 mutants labelled with ³H-palmitic acid and immunoprecipitated with anti-GFP. Graph bars correspond to the mean ± SEM ³H-palmitic acid incorporation normalised to WT construct in three to four independent experiments. (E) Normalised mean fura-2 responses evoked by Ca²⁺ readmission in HEK-TKO cells transiently transfected with the indicated ORAI1-YFP constructs and pre-exposed to Tg 1 µM for 8 min. (F) Area under the curve of the responses in E. Data are mean ± SEM of eight independent experiments. One-way ANOVA Dunnett’s multiple comparisons test.

Figure 1—figure supplement 1. Right, Orai1 protein sequences aligned with CLustalW for the indicated organisms.

Cysteines susceptible to be S-acylated are highlighted in yellow. Left, Schematic ORAI1 representation. Superimposed structures of the WT and H206A dOrai (PDB ID: 4HKR and 6BBF) conformations in ribbon representation highlighting cysteine residues at position 126, 143, and 195.

Figure 1—figure supplement 2. Top; fluorescence images of WT O1/S1 cells (left) and averaged mCh-STIM1 and ORAI1-YFP fluorescence of the different O1/S1 stable cell lines Data are mean ± SE of 53–132 cells.

Bottom; Averaged fura-2 responses (left) and integrated response (right) of O1/S1 cells bearing or not the indicated ORAI1 mutation(s). Data are mean ± SEM of 44–82 cells from two independent experiments. One-way ANOVA Dunnett’s multiple comparisons test.