Ji-Won Huh^a,1, Takuma Shibata^b,1, Misun Hwang^a, Eun-Hye Kwon^a, Min Seong Jang^a,c, Ryutaro Fukui^b, Atsuo Kanno^b,d, Da-Jung Jung^a, Myoung Ho Jang^a,c,2, Kensuke Miyake^b,d,2, and You-Me Kim^a,e,2

^aDivision of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea;
^bDivision of Innate Immunity, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108 8639, Japan;
^cAcademy of Immunology and Microbiology, Institute for Basic Science, Pohang 790-784, Republic of Korea;
^dLaboratory of Innate Immunity, Center for Experimental Medicine and Systems Biology, Institute of Medical Science, University of Tokyo, Tokyo 108 8639, Japan; and
^eDepartment of Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea

Abstract
The proper trafficking and localization of Toll-like receptors (TLRs) are important for specific ligand recognition and efficient signal transduction. The TLRs sensing bacterial membrane components are expressed on the cell surface and recruit signaling adaptors to the plasma membrane upon stimulation. On the contrary, the nucleotide-sensing TLRs are mostly found inside cells and signal from the endolysosomes in an acidic pH-dependent manner. Trafficking of the nucleotide-sensing TLRs from the endoplasmic reticulum to the endolysosomes strictly depends on UNC93B1, and their signaling is completely abolished in the 3d mutant mice bearing the H412R mutation of UNC93B1. In contrast, UNC93B1 was considered to have no role for the cell surface-localized TLRs and signaling via TLR1, TLR2, TLR4, and TLR6 is normal in the 3d mice. Unexpectedly, we discovered that TLR5, a cell surface receptor for bacterial protein flagellin, also requires UNC93B1 for plasma membrane localization and signaling. TLR5 physically interacts with UNC93B1, and the cells from the 3d or UNC93B1-deficient mice not only lack TLR5 at the plasma membrane but also fail to secret cytokines and to up-regulate costimulatory molecules upon flagellin stimulation, demonstrating the essential role of UNC93B1 in TLR5 signaling. Our study reveals that the role of UNC93B1 is not limited to the TLRs signaling from the endolysosomes and compels the further probing of the mechanisms underlying the UNC93B1-assisted differential targeting of TLRs.

Toll-like receptor trafficking, 3d

¹J.-W.H. and T.S. contributed equally to this work.
²To whom correspondence may be addressed.

Author contributions: T.S., M.H.J., K.M., and Y.-M.K. designed research; J.-W.H., T.S., M.H., E.-H.K., M.S.J., R.F., A.K., and D.-J.J. performed research; J.-W.H., T.S., M.H., E.-H.K., M.S.J., R.F., A.K., D.-J.J., M.H.J., K.M., and Y.-M.K. analyzed data; and J.-W.H. and Y.-M.K. wrote the paper.