Development of palmitoylation-inducing protein tag causing membrane targeting

Abstract

Protein S-palmitoylation is a reversible post-translational modification that alters the localization, stability, and function of hundreds of proteins in the cell. In the eukaryote system, many proteins involved in biological functions such as signaling transduction are regulated by palmitoylation. In this study, based on the fact that palmitoylation can alter the subcellular localization of proteins, I aimed to develop a tool that could exploit these properties by inducing ectopic palmitoylation in proteins that were not originally palmitoylated. Using EGFP as a readout, I visualized palmitoylation-induced changes in the subcellular localization of proteins. In order to induce palmitoylation of EGFP, I generated the Palmito-tag, a novel and simple synthetic amino acid sequence. Incorporation of the Palmito-tag in EGFP successfully induced EGFP localization in the plasma membrane and extracellular vesicles, thereby demonstrating that Palmito-tag functions as a de facto modulator for subcellular localization of a target protein. Of note, Palmito-tag appeared to induce membrane localization of EGFP with a much higher efficiency than previously reported palmitoylation-prone sequence of GAP43. Collectively, These results demonstrate that protein S-palmitoylation appears to play a direct role in regulating the subcellular localization of proteins, especially membrane targeting. Moreover, Palmito-tags developed through this study can be used sufficiently to impart membrane targeting property to proteins by inducing palmitoylation. Considering the previous reports that subcellular localization of a palmitoylated protein is usually determined by the identity of DHHC which mediates the palmitoylation, and the specific target sequence of the DHHC has not yet been identified, it is plausible that the Palmito-tag sequence may represent an effective target sequence for DHHC which mediates plasma membrane localization of a target protein. Beyond that, my data suggest that Palmito-tag could also be applied for drug delivery for therapeutic purposes.