Charge-dependent localization of Toll-like receptor 5 at the plasma membrane

Proper subcellular localization of Toll-like receptors (TLRs) is essential for initiating appropriate innate immune responses against pathogens while avoiding self-reactivity. Unc-93 homolog B1 (UNC93B1) is known to mediate the intracellular trafficking of nucleotide-sensing TLRs such as TLR9 which undergoes rapid internalization into endolysosomes upon reaching the cell surface. We previously demonstrated that UNC93B1 also facilitates the plasma membrane localization of TLR5, a sensor for bacterial flagellin. Unlike TLR9, TLR5 remained stably at the cell surface under steady-state conditions, suggesting the involvement of distinct sorting mechanisms. Using mutagenesis-based approaches, we found that the cytoplasmic domain of TLR5 is required for its surface retention, whereas the cytoplasmic domain of TLR9 is dispensable for internalization. Notably, TLR5 contains polybasic residues in its C-terminal region, absent in other TLRs. Deletion or alanine substitution of these residues led to constitutive endocytosis of TLR5. Conversely, appending the TLR5 C-terminal region to the C-terminus of TLR9 promoted its surface accumulation. Moreover, when the TLR5 C-terminal sequence was fused to a cytosolic protein along with a myristoylation motif, it mediated membrane association of the cytosolic protein in a charge-dependent manner. We further found that this region can directly interact with phosphatidic acid, an anionic phospholipid enriched in the plasma membrane. These findings reveal an electrostatic mechanism by which TLR5 is selectively retained at the plasma membrane, providing new insight into receptor-specific localization of TLRs.