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.

Commercial anti-GM130 antibody was purchased from BD Transduction (San Jose, CA). The monoclonal anti-FLAG epitope (M2), the polyclonal anti-FLAG and the anti-b-actin (clone AC-74) were obtained from Sigma (Oakville, Ontario, Canada). The c-Myc (9E10) monoclonal antibodies, as well as the polyclonal p115 (H-300) and TRAF3 (C-20, H-20, and G-6) antibodies were purchased from Santa Cruz (Santa Cruz, CA). The anti-GFP (monoclonal 1218) antibody and the polyclonal goat anti-GFP antibody were obtained from ABCAM (Cambridge, MA) and US Biological (Swampscott, MA) respectively. The anti-ERGIC53 and anti-calnexin antibodies were from Enzo Life Sciences, anti-p-IRF3 Ser398 was from Millipore (Billerica, MA) and anti-ISG54 was from Novus Biologicals (Littleton, CO). The polyclonal anti-Sec16A and p115 antibodies were obtained from Bethyl Laboratories and Santa Cruz. The plasmid encoding for EGFP-Sec16A was a kind gift of Dr. David Stephens (University of Bristol, UK). Human TRAF3 and p115 cDNAs were amplified from the MGC bank collection and respectively subcloned in pcDNA3 and pTag2B (FLAG) or pTag3B (Myc) vectors (Invitrogen, Burlington, ON, Canada). Human TRAF6 cDNAs were purchased from Origene (Rockville, MD) and subcloned in the pTag2B/3B vectors. The pFLAG-CMV2-TBK1 and pFLAG-TRAF3 Y440/ Q442A were gifts from Drs. John Hiscott (McGill University). pcDNA3.1-FLAG-MAVS construct was from Rongtuan Lin (McGill University). The pcDNA3-His-TRIF construct was from Dr. Daniel Lamarre (Université de Montréal). The pRK5-TRAF2-FLAG was obtained from Dr. Nathalie Grandvaux (Université de Montréal). pFLAG-CMV2 TRAF3 deletion mutants (1-117, 1-381, 114-568, 259-568 and 389-568) were from Dr. Carl Ware (La Jolla Institute for Allergy and Immunology). The IFNß reporter plasmid, pGL3-IFN-ß-LUC was described previously [77] as well as the ISG56-luciferase [78] and the NF-kB p2(2)TK reporter plasmids [77] . The pFLAG-TRAF3 mutant with C-terminal retention motif AKKFF was generated by PCR and subcloned in pCDNA3.1 (+) and pMRX-ires-puro (a kind gift from Dr. Shoji Yamaoka, Tokyo Medical and Dental University, Figure 11 . Through its ability to interact and colocalize with components of the ERES (Sec16A, depicted as thick green lines), ERGIC (ERGIC53 and p115) and the cis-Golgi apparatus (p115 and GM130), a subpopulation of TRAF3 (red circles) resides in the ER-to-Golgi vesicular compartment in non-infected cells. Upon dsRNA and dsDNA sensing, the cis-Golgi disorganizes into punctate structures, giving rise to membrane-bound compartments composed of at least GM130 and TRAF3 (dashed line). We propose that these membrane-bound compartments allow the proper positioning of TRAF3 with MAVS at Mitochondrial-Associated endoplasmic reticulum Membranes (MAM) [86] . There, being in close proximity with a component of the exocyst (sec5) and the translocon (Sec61b), TRAF3 allows the activation of TBK1 and IRF3 leading to activation of the type I IFN response. A similar scenario was recently proposed for STING (yellow circles) where in response to DNA virus infection, it traffics from the ER to the cis-Golgi apparatus and finally to a distinct perinuclear region for the activation of TBK1 [61, 62] . MTOC: microtubuleorganizing center. doi:10.1371/journal.ppat.1002747.g011 Japan). Poly I:C was purchased from GE HealthCare (Waukesha, WI) and transfected with Lipofectamine2000 (Invitrogen) at final concentrations of 1.0 to 2.5 mg/ml. Poly dA:dT was from InvivoGen and used at 1