Proteomic Profiling of the TRAF3 Interactome Network Reveals a New Role for the ER-to-Golgi Transport Compartments in Innate Immunity

van Zuylen, Wendy J.; Doyon, Priscilla; Clément, Jean-François; Khan, Kashif Aziz; D'Ambrosio, Lisa M.; Dô, Florence; St-Amant-Verret, Myriam; Wissanji, Tasheen; Emery, Gregory; Gingras, Anne-Claude; Meloche, Sylvain; Servant, Marc J.

Herein, we report that TRAF3 localizes to the ER-to-Golgi compartments through its ability to interact with p115-and Sec16A-containing complexes. A pharmacological approach using the microtubule depolarizing agent nocodazole led to the redistribution of TRAF3 into small punctate cytoplasmic structures discrete from the ER along with both Golgi matrix proteins p115 and GM130. Both the structure and positioning of the Golgi apparatus have been shown to be highly dependent on the microtubule cytoskeleton [57] . Interestingly, a link between TRAF3 and the microtubule network has been already established in a previous study through its interaction with Microtubule-Interacting Protein that associates with TRAF3 (MIP-T3) [58] . TRAF3 was dissociated from this complex upon CD40L stimulation and, consequently, it was suggested that microtubule association of TRAF3 could be responsible for directing TRAF3 to defined membrane microdomains in the cell. A similar scenario is proposed here where, in response to viral infection, the association of TRAF3 with complexes containing p115 and Sec16A at the ER-to-Golgi vesicular pathway may play an important role in positioning TRAF3 with MAVS (see Figure 11 ). Indeed, the following findings suggests a role for Sec16A and p115 in the TRAF3-mediated RLH type I IFN response: (1) Sec16A and p115 are found in immunocomplexes containing TRAF3, but not TRAF2 or TRAF6; (2) inactivation of TRAF3 by deletion of its Nterminal RING finger domain and the C-terminal TRAF domain displaces TRAF3 from the ER-Golgi transport compartments; (3) in non-treated cells, TRAF3 colocalizes and tightly associates with p115, Sec16A, ERGIC53 and GM130, markers of the ER-to-Golgi vesicular compartment; (4) activation of the RLH pathway leads to reorganization of the Golgi apparatus into punctate structures containing TRAF3 and GM130; (5) an increased association between TRAF3, Sec16A, p115 and TBK1 is observed in virally-infected, dsRNA-and dsDNA-transfected cells; (6) mild overexpression of both proteins enhances SeV-, TBK1-and MAVS-stimulated IFNb, ISG56 and NF-kB promoter induction; (7) knocking down the expression level of p115 or Sec16A affects the cellular distribution of TRAF3, impairs its capacity to associate with MAVS and diminishes the type I IFN response following poly I:C or polydA:dT transfection and SeV infection; and (8) enforced retention of TRAF3 at the ER-to-Golgi compartment by the addition of a COPI and COP II sorting signal peptide impairs TRAF3 recruitment to the cis-Golgi and diminishes the type I IFN response. Thus, we propose that these two trafficking proteins, Sec16A and p115, form a complex with TRAF3 at ER-to-Golgi transport compartments in order to ensure its proper recruitment to the mitochondrial network during a viral infection. Interestingly, enforced expression of Sec16A or p115 also increases TRIFmediated IFNb promoter activation, reinforcing the role for the ER-to-Golgi vesicular compartment in TLR3 and TLR4 signalling, as recently reviewed [59] .