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.

TRAF3 localization and interaction with components of the ER-to-Golgi vesicular pathway requires its protein integrity It has been proposed that a structurally intact TRAF3 molecule is required for its biological function. Indeed, TRAF3 lacking its N-terminal RING or the C-terminal TRAF domain lacks antiviral activity [20] . We therefore examined the subcellular localization of TRAF3 deletion mutants in reconstituted TRAF3 knockout MEF cells. Removal of the N-terminal Ring Finger domain ( Figure 3A , panel 1), the N-terminal Ring and Zinc finger domains ( Figure 3A , panel 2) or the C-terminal TRAF domain ( Figure 3A , panel 3) resulted in TRAF3 molecules that no longer colocalize with the Golgi marker GM130. Furthermore, coimmunoprecipitation experiments in 293T cells revealed that immunocomplexes containing p115 are detected only with full length TRAF3 and that Sec16A-containing immunocomplexes required at least the isoleucine zipper and the TRAF domain ( Figure 3B ). Moreover, TRAF3 is known to interact with several substrates containing a particular motif (PxQxS/T) called the TRAF interaction motif (TIM) [45] . The mutation of two amino acids located in the TIMbinding pocket of TRAF3, Y440 and Q442, abrogates these interactions [20] . Interestingly, a strong interaction was detected between FLAG-TRAF3 Y440/Q442A and Myc-p115 or EGFP-Sec16A ( Figures 3C and 3D ), implying that this interaction is independent of the TIM motif. Thus, it is not clear yet whether TRAF3 interacts directly with Sec16A or p115 or requires other components such as TFG ( [46] and see Figure S1 ). Collectively, these data suggest that an intact TRAF3 molecule is required for its proper localization and interaction with components of the ERto-Golgi vesicular pathway.