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.

In order to find novel players involved in the type I IFN pathway, we have used a functional proteomics approach based on FLAG affinity purification and mass spectrometry analysis (AP/ MS). HEK293 cells stably expressing FLAG-TRAF3 were harvested, subjected to IP with an anti-FLAG antibody under native conditions and FLAG-TRAF3 complexes were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). In parallel, multiple AP/MS analyses were performed from cells expressing the FLAG alone. Following standard database searches, stringent statistical filtering was performed using SAINT (see Methods). Proteins detected with AvgP$0.7 were manually inspected for frequency of detection across a database of ,1000 AP/MS analyses, and proteins frequently detected in AP-MS experiments were removed. This resulted in the identification of 12 interaction partners for TRAF3, including TBK1, a well-known TRAF3 interactor [12, 13] . Surprisingly, Sec16A and p115, two proteins involved in ER-to-Golgi vesicular trafficking, were found to associate with TRAF3 immunocomplexes with a high confidence ( Figure 1A and Figure S1 ).