The effects of chloride ion binding on the photochemical properties of sensory rhodopsin II from Natronomonas pharaonis.

Whether Cl(-) binds to the sensory rhodopsin II from Natronomonas pharaonis (NpSRII) that acts as a negative phototaxis receptor remains controversial. Two previous photoelectrochemical studies using SnO2 transparent electrodes and ATR-FTIR demonstrated that Cl(-) binding affects the photoinduced proton release from Asp193 in phospholipid (PC)-reconstituted NpSRII (Iwamoto et al., 2004; Kitade et al., 2009). In this study, we investigated the effects of Cl(-) on the photochemistry of NpSRII solubilized by detergent (DDM). Even under these conditions, Cl(-) could bind to NpSRII with a Kd of approximately 250 mM; this value is ∼ 10-fold larger than that in the PC membrane. The binding of Cl(-) to NpSRII depended on the pH of the medium. In addition, Cl(-) binding induced the following effects: (1) a small red shift in the absorbance spectrum originating from the partial protonation of Asp75, (2) the formation of an interaction through a hydrogen-bonding network between Asp75 and Asp193, which is a proton-releasing residue, (3) several changes of the kinetic behavior of the photocycle, and (4) a photoinduced initial proton release from Asp193. The pKa values of Asp193 at various Cl(-) concentrations were also estimated. Based on the difference between the pKa values of Asp193 in Cl(-) bound and unbound NpSRII, the distance between the bound Cl(-) and Asp193 was determined to be approximately 6.1 Å, which agrees with the value estimated from the crystal structure presented by Royant et al. (2001). Therefore, the Cl(-) binding site affecting the photochemical properties of NpSRII is identical to the site proposed by Royant et al. (2001). This assignment was also supported by an experiment that introduced a mutation at Arg72.

UNC93B1 physically associates with human TLR8 and regulates TLR8-mediated signaling.

Toll-like receptors (TLRs) 3, 7, 8, and 9 are localized to intracellular compartments where they encounter foreign or self nucleic acids and activate innate and adaptive immune responses. The endoplasmic reticulum (ER)-resident membrane protein, UNC93B1, is essential for intracellular trafficking and endolysosomal targeting of TLR7 and TLR9. TLR8 is phylogenetically and structurally related to TLR7 and TLR9, but little is known about its localization or function. In this study, we demonstrate that TLR8 localized to the early endosome and the ER but not to the late endosome or lysosome in human monocytes and HeLa transfectants. UNC93B1 physically associated with human TLR8, similar to TLRs 3, 7, and 9, and played a critical role in TLR8-mediated signaling. Localization analyses of TLR8 tail-truncated mutants revealed that the transmembrane domain and the Toll/interleukin-1 receptor domain were required for proper targeting of TLR8 to the early endosome. Hence, although UNC93B1 participates in intracellular trafficking and signaling for all nucleotide-sensing TLRs, the mode of regulation of TLR localization differs for each TLR.