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.

To further validate the interaction between TRAF3 and these new interactors, we next analyzed their subcellular localization by confocal microscopy. The vesicle-tethering protein p115 is known to colocalize and interact specifically with the NH2 terminus of the cis-Golgi protein GM130 ( [36] and see Figure 2A , panel 2). Upon ectopic expression of Myc-p115 and FLAG-TRAF3, we observed a co-localization of these two proteins (Figure 2A, panel 1) . FLAG-TRAF3 was also observed to localize to the Golgi apparatus where it exhibits a high degree of overlap with the cis-Golgi marker GM130 (Figure 2A, panel 3) . p115 was previously reported to be present in the ERGIC, through an interaction involving activated Rab1 [37, 38] . This cellular localization of FLAG-p115 can be visualized with the conventional ERGIC marker, ERGIC53 (Figure 2A panel 4) . Notably, FLAG-TRAF3 (or Myc-TRAF3 (unpublished data)) was also present in the ERGIC (Figure 2A , panel 5). No significant colocalization was detected between TRAF3 and the ER marker calnexin (Figure 2A In HeLa cells, Sec16A was demonstrated to define ERES [24, 25] , localizing to punctate structures on the ER membrane. This pattern was reproduced in this study ( Figure 2B , panel 1). Since Rab1 recruitment of p115 to ERES [26] represents an essential step for the subsequent docking of ER-derived vesicles to the ERGIC [39] , we next examined the colocalization of EGFP-Sec16A and FLAG-p115. The two proteins clearly colocalized at the perinuclear region ( Figure 2B , panel 2). FLAG-TRAF3 and EGFP-Sec16A also mainly colocalized at the perinuclear region in HeLa cells ( Figure 2B, panel 3) . Moreover, a colocalization of FLAG-TRAF3 with endogenous Sec16A at ERES distributed in the cytoplasm could be observed. However, some FLAG-TRAF3 punctae also appeared in close proximity to those containing Sec16A ( Figure 2B , panel 4, compare arrows). Ectopic expression of FLAG-TRAF2 and FLAG-TRAF6 revealed that only TRAF3 exhibits a cellular Golgi-like distribution ( Figure S2A ) and colocalizes with endogenous Sec16A ( Figure S2B ) or Myc-p115 ( Figure S2C ). Importantly, endogenous staining of TRAF3 revealed that the majority of TRAF3 proteins localized to the juxtanuclear region containing both the cis-Golgi marker GM130 and the ERGIC marker ERGIC53 ( Figure 2C ).