A Novel Synthetic Tag Induces Palmitoylation and Directs the Subcellular Localization of Target Proteins

Abstract

Proper subcellular localization is essential to exert the designated function of a protein, not only for endogenous proteins but also transgene-encoded proteins. Post-translational modification is a frequently used method to regulate the subcellular localization of a specific protein. While there are a number of tags that are widely used to direct the target protein to a specific location within a cell, these tags often fail to emulate the dynamics of protein trafficking, necessitating an alternative approach to the direct subcellular localization of transgene-encoded proteins. Here, we report the development of a new synthetic polypeptide protein tag comprised of ten amino acids, which promotes membrane localization of a target protein. This short synthetic peptide tag, named “Palmito-Tag”, induces ectopic palmitoylation on the cysteine residue within the tag, thereby promoting membrane localization of the target proteins without affecting their innate function. We show that the target proteins with the Palmito-Tag are incorporated into the membranous organelles within the cells, including the endosomes, as well as extracellular vesicles. Given the reversible nature of palmitoylation, the Palmito-Tag may allow us to shift the subcellular localization of the target protein in a context-dependent manner. With the advent of therapeutic applications of exosomes and other extracellular vesicles, we believe that the ability to reversibly modify a target protein and direct its deposition to the specific subcellular milieu will help us explore more effective venues to harness the potential of extracellular vesicle-based therapies. © 2025 Elsevier B.V., All rights reserved.