Anterograde trafficking of Toll-like receptors requires the cargo sorting adaptors TMED-2 and 7

Abstract

Toll-Like Receptors (TLRs) play a pivotal role in immunity by recognising conserved structural features of pathogens and initiating the innate immune response. TLR signalling is subject to complex regulation that remains poorly understood. Here we show that two small type I transmembrane receptors, TMED2 and 7, that function as cargo sorting adaptors in the early secretory pathway are required for transport of TLRs from the ER to Golgi. Protein interaction studies reveal that TMED7 interacts with TLR2, TLR4 and TLR5 but not with TLR3 and TLR9. On the other hand, TMED2 interacts with TLR2, TLR4 and TLR3. Dominant negative forms of TMED7 suppress the export of cell surface TLRs from the ER to the Golgi. By contrast TMED2 is required for the ER-export of both plasma membrane and endosomal TLRs. Together, these findings suggest that association of TMED2 and TMED7 with TLRs facilitates anterograde transport from the ER to the Golgi.

Keywords: COP-coated vesicles; TLRs; endoplasmic reticulum; p24; protein transport.

FIGURE 1

TMED2 interacts with TMED7. Proximity Ligand Assays (PLA) in HEK293T cells and murine BMDM enable visualisation of protein–protein interactions. Cells were transiently co‐transfected and fixed prior to PLA staining and analysis by confocal microscopy. The fluorescent PLA signal was visualised in the red (559 nm) channel. The nucleus, stained with DAPI, was visualised in the blue (405 nm) channel, while images of cell structures were obtained using DIC microscopy. The overlay shows a merged, pseudo‐coloured images of the three images collected with the above listed channels. Images are representatives of three independent experiments. PLA signals were produced in HEK293T cells and BMDM co‐transfected with TMED2‐HA and TMED7‐FLAG, and HEK293T cells co‐transfected with CC‐FLAG (TMED7 ectodomain) and TMED2‐HA. No PLA signal was detected in cells transfected with either TMED2‐HA, TMED7‐FLAG or CC‐FLAG.

FIGURE 2

TMED7 Interacts with membrane TLRs and TMED2 with both endosomal and membrane. (A, B) HEK293T cells were transiently transfected with the plasmids listed. Following cell lysis, the whole cell lysates were either prepared for analysis or incubated with M2 anti‐FLAG beads to immunoprecipitate protein (IP) with FLAG‐tags. Whole lysate (WL) and IP were resolved by SDS‐PAGE and analysed by western blot using immunoblotting (IB). (C, D) HEK293T cells were transiently co‐transfected with plasmids listed and fixed prior to PLA staining and analysis by confocal microscopy. The PLA signal was visualised in the red (559 nm) channel. The nucleus stained with DAPI was visualised in the blue (405 nm) channel. Images of cell structures where obtain using DIC microscopy. The overlay shows a merged, pseudo‐coloured images of the three images collected with the above listed channels. Images are representatives of three independent experiments. Scale bar 5 or 10 μm.

FIGURE 3

TMED7 Affects the ER export of some TLRs. Triple‐colour imaging of HEK293T cells transiently expressing, (A) TMED7‐mCherry or TMED7T‐mCherry, (B) TLR2‐EYFP, TLR4‐Citrine and MD2, or TLR3‐EYFP, (C) TLR2‐EYFP and TMED7‐mCherry or TMED7T‐mCherry, (D) TLR4‐citrine and TMED7‐mCherry or TMED7T‐mCherry, E: TLR3‐EYFP and TMED7‐mCherry or TMED7T‐mCherry. TMED7 constructs were visualised in the red (559 nm) channel and TLR constructs in the yellow (515 nm) channel. The ER was immunostained and visualised in the far‐red (635 nm) channel, while the Golgi in the blue (405 nm) channel. The overlay is a merge of the red channel with either the far‐red or the blue channel respectively. Quantification of the Pearson's coefficient (R) for the dual colour merged imaged. Images are representatives of three independent experiments. Panel A and D: scale bar 5 μm; panel E; scale bar 8 μm; panel B and C: scale bar 10 μm.

FIGURE 4

TMED2 regulates the ER Export of some TLRs. Triple‐colour imaging of HEK293T cells transiently expressing, (A) TMED2‐mCherry or TMED2T‐mCherry; (B) TLR2‐EYFP and TMED2‐mCherry or TMED2T‐mCherry; (C) TLR4‐citrine and TMED2‐mCherry or TMED2T‐mCherry, (D) TLR3‐EYFP and TMED2‐mCherry or TMED2T‐mCherry. TMED2 constructs were visualised in the red (559 nm) channel and TLR constructs in the yellow (515 nm) channel. The ER was immunostained and visualised in the far‐red (635 nm) channel, while the Golgi in the blue (405 nm) channel. The overlay is a merge of the red channel with either the far‐red or the blue channel respectively. Quantification of the Pearson's coefficient (R) for the dual colour merged imaged. Images are representatives of three independent experiments. Scale bar 10 μm.