Figure 2. UNC93B1 controls ER exit of TLRs 3, 7, 9, 11, and 13.
(A) TLR9 fails to exit the ER in cells lacking functional UNC93B1. Lysates from 3d iMac cells complemented with either UNC93B1-WT or UNC93B1-H412R and expressing TLR9-HA were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. The precursor (black arrow), ER (white arrow) and cleaved (grey arrow) forms of TLR9-HA are indicated. (B) UNC93B1 is required for TLR9 loading into COPII vesicles. RAW264 macrophages stably transduced with retroviruses encoding control or Unc93b1-directed shRNA and expressing TLR9-HA were used in an in vitro COPII budding assay as described in (Figure 1H). Lysates of purified vesicles or donor membranes were probed with the indicated antibodies. (C) The transmembrane and cytosolic domain of TLR9 is sufficient to confer UNC93B1-dependence. (Left) schematic of TLR9 and the CD4-TLR9 chimera. Transmembrane (TM), ectodomain (Ecto) and cytosolic domain (Cyto) are indicated. (Right) CD4-TLR9 was expressed in HEK293Ts together with FLAG-tagged UNC93B1-WT or UNC93B1-H412R. Total lysates were analyzed by SDS-PAGE and immunoblotted with anti-CD4 and anti-FLAG antibodies. EndoH-sensitive (white arrow) and resistant (black arrow) forms are indicated. (D) CD4 trafficking to the cell surface is normal in Unc93b13d/3d cells. Splenocytes from C57BL/6 (blue line) or Unc93b13d/3d (red line) mice were stained with anti-CD4 and analyzed by flow cytometry. (E) CD4-TLR chimeric proteins for each of the indicated TLRs were expressed in HEK293Ts together with FLAG-tagged UNC93B1-WT or UNC93B1-H412R. Lysates were separated by SDS-PAGE and visualized by immunoblot with anti-HA and anti-FLAG antibodies. EndoH-sensitive (white arrows) and resistant (black arrows) forms are indicated. The chimeras were constructed as shown in Figure 1E, except with the addition of a C-terminal HA tag. Results are representative of at least three experiments (A, C, and E) or two experiments (B and D).