Homeostatic regulation of STING by retrograde membrane traffic to the ER

Abstract

Coat protein complex I (COP-I) mediates the retrograde transport from the Golgi apparatus to the endoplasmic reticulum (ER). Mutation of the COPA gene, encoding one of the COP-I subunits (α-COP), causes an immune dysregulatory disease known as COPA syndrome. The molecular mechanism by which the impaired retrograde transport results in autoinflammation remains poorly understood. Here we report that STING, an innate immunity protein, is a cargo of the retrograde membrane transport. In the presence of the disease-causative α-COP variants, STING cannot be retrieved back to the ER from the Golgi. The forced Golgi residency of STING results in the cGAS-independent and palmitoylation-dependent activation of the STING downstream signaling pathway. Surf4, a protein that circulates between the ER/ ER-Golgi intermediate compartment/ Golgi, binds STING and α-COP, and mediates the retrograde transport of STING to the ER. The STING/Surf4/α-COP complex is disrupted in the presence of the disease-causative α-COP variant. We also find that the STING ligand cGAMP impairs the formation of the STING/Surf4/α-COP complex. Our results suggest a homeostatic regulation of STING at the resting state by retrograde membrane traffic and provide insights into the pathogenesis of COPA syndrome.

Fig. 1. The α-COP variants activate the STING pathway.

a HEK293T cells were transfected as indicated, together with an ISRE (also known as PRDIII or IRF-E)-luciferase reporter. Luciferase activity was then measured. Data represent mean s.e.m. of three independent experiments. b STING and/or α-COP were stably expressed in Sting^−/− MEFs. Cell lysates were prepared and analyzed by western blot. c Quantitative real-time PCR (qRT-PCR) of the expression of innate immune genes in MEFs expressing the α-COP variants. The indicated gene expression was normalized on the basis of GAPDH content and the log₂ fold change compared to α-COP (WT) was plotted as a heatmap. See also Supplementary Fig. 3.

Fig. 2. The α-COP variants alter STING localization to the Golgi.

a, b α-COP and EGFP-STING were stably expressed in Sting^−/− MEFs as indicated. Cells were fixed, permeabilized, and stained for TGN38 (a Golgi protein) (a) or p-TBK1 (b). Nuclei were stained with DAPI (blue). Color scales and intensity levels are indicated below images. Scale bars, 10 µm. c α-COP-FLAG (WT) or α-COP-FLAG (E241K) was stably expressed in EGFP-STING-reconstituted MEFs. Cells were fixed and processed for ultrathin cryosections. They were immunostained with anti-GFP (rabbit) and anti-FLAG (mouse) antibodies. As secondary antibodies, colloidal gold particle-conjugated donkey anti-rabbit antibody (12 nm) and anti-mouse (6 nm) were used. Arrowheads indicate α-COP signal, red arrows indicate GFP signal on the Golgi, and blue arrows indicate GFP signal that was not associated with the Golgi. Go, the Golgi stack. Scale bars, 500 nm. d Cell lysates were prepared from MEFs expressing various α-COP as indicated, and α-COP was immunoprecipitated with anti-FLAG antibody. Cell lysates and the immunoprecipitates were analyzed by western blot.

Fig. 3. Surf4 binds STING and α-COP, and is required for STING localization to the ER.

a, b MEFs expressing EGFP-STING were treated with the indicated siRNA for 48 h. Cells were fixed, permeabilized, and stained for GM130 and TGN38. Color scales and intensity levels are indicated below images. Scale bars, 10 µm. c Sting^−/− MEFs or Sting^−/− MEFs reconstituted of EGFP-STING were treated with the indicated siRNA. Cell lysates were prepared and analyzed by western blot. d, e HEK293T cells were transfected with the indicated plasmids. Cell lysates were prepared and α-COP-FLAG was immunoprecipitated. Cell lysates and the immunoprecipitates were analyzed by western blot.

Fig. 4. STING activation with the α-COP variants requires the ER-to-Golgi traffic and palmitoylation of STING, but not cGAS.

a, b MEFs expressing various α-COP as indicated were treated with BFA (0.3 µg/ml) for 8 h (a) or with C178 (10 µM) for 8 h (b). Cell lysates were prepared and analyzed by western blot. c α-COP was stably expressed in cGAS-KO MEFs. Cell lysates were prepared and analyzed by western blot. d A model of STING regulation by membrane traffic between the ER and the Golgi. e, f α-COP-FLAG was stably expressed WT MEFs, or HA-Surf4 was stably expressed in Sting^-/- MEFs that were reconstituted of EGFP-STING. Cells were stimulated with cGAMP (e) or DMXAA (f) for 1 h. Cell lysates were prepared, and α-COP-FLAG or EGFP-STING was immunoprecipitated. Cell lysates and the immunoprecipitates were analyzed by western blot. g HEK293T cells were transfected with the indicated plasmids. Cell lysates were prepared and EGFP-STING were immunoprecipitated. Cell lysates and the immunoprecipitates were analyzed by western blot.