The Role of S-Acylation in the Regulation of Store-Operated Calcium Entry

Abstract

Store-operated calcium entry (SOCE) is critical for T cell-mediated immunity, specifically, for T cell activation, clonal expansion, and differentiation. In T cells, SOCE is facilitated by the plasma membrane (PM) localized protein Orai1 and the endoplasmic reticulum (ER) membrane localized protein STIM1. Upon T cell receptor activation, ER calcium stores are depleted, leading to STIM1 activation, which undergoes a conformational change, allowing it to interact with Orai1 at ER/PM junctions. This interaction enables Orai1 gating, leading to calcium entry into the cell. Although the importance of Orai1 and STIM1 in SOCE has been established, it is still unclear what mechanisms regulate the formation of Orai1/STIM1 complexes at ER/PM junctions. We have found that, among several other regulatory T cell proteins, both Orai1 and STIM1 are S-acylated. S-acylation is the post-translational lipidation of a cysteine residue via a labile thioester bond. Using biochemical, electrophysiological, and advanced imaging approaches, we have found that S-acylation of Orai1 and STIM1 is critical for their function. Using acyl-biotin exchange, we determined not only are Orai1 and STIM1 S-acylated, but also the S-acylation of Orai1 is rapid, transient, and calcium-dependent. To further study the importance of S-acylation on SOCE, we used whole cell recording to show that the S-acylation of both Orai1 and STIM1 is important for proper calcium channel currents. Lastly, after performing total internal reflection fluorescence, Förster resonance energy transfer, and Fura-2 calcium imaging, we determined acylation-deficient Orai1 significantly decreases SOCE and does not efficiently interact with STIM1 to form functional calcium channels. This work describes a previously unknown signaling pathway that regulates SOCE in T cells, and contributes to T cell-mediated immunity.