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.

Statistical analyses were performed using GraphPad Prism version 5.0 for Mac (GraphPad Software, San Diego, CA). Comparison of two groups was carried out using a two-tailed unpaired t-test, and comparison of more than two groups was carried out with one-way ANOVA and a Bonferroni posttest. Statistical significance was accepted at a P-value below 0.05. Figure S1 The TRAF3 interactome network. (A) AP-MS data with $0.5 AvgP SAINT value. Indicated baits and prey (HUGO gene names; USO1 is the gene name for p115) are listed, alongside the accession number (protein NCBI gi) for the prey, and SAINT output data. Columns are as follows («|» is a delimiter for biological replicates): «IP» are unique identifiers for the experiment in the ProHits database; «Spec» are the spectral counts in each individual experiment; «SpecSum» is the sum of the spectral counts across all analyses. «iProb» is the initial probability in an individual experiment; «crtlCounts» are the spectral counts in five virtual controls, as defined in Methods; «AvgP» is the average of the individual probabilities. The following proteins passed the SAINT threshold filter but were excluded from further analysis, based on high-frequency of detection in FLAG AP/MS analysis from HEK293 cells: EWSR1, FUS, HNRNPC, TUBB2C, HSPA9, HNRNPM, HNRNPH1, ABCA13 and CEP290. (B) Overlay of the filtered mass spectrometry data with literaturecurated interactions (as reported in BioGRID version 3.1.76*). Data is visualized in Cytoscape**. The blue colored edges are from the mass spectrometry data in Figure S1A ; the grey from literature-curated interactions. The thickness of the blue edges corresponds to the number of spectral counts for each of the proteins. Dashed lines on the BioGRID data are for ''yeast two hybrid'', ''colocalization'' or ''enzymatic activity'' annotations in BioGRID; continuous lines are for co-IP coupled to mass spectrometry or to immunoblotting, as well as for co-crystal structures. The two baits, TRAF3 and USO1/p115 are shown as larger nodes. The previously known TRAF3 interactor is shown in pink. New TRAF3 interactors USO1/p115 and SEC16A are shown in orange. * Stark C, Breitkreutz BJ, Chatr-Aryamontri A, Boucher L, Oughtred R, Livstone MS, Nixon J, Van Auken K, Wang X, Shi X, Reguly T, Rust Figure S5 COPI/COPII-vesicular retention of the TRAF3-AKKFF mutant affects its extraction efficiency as well as interaction with MAVS. 293T cells were co-transfected with Myc-MAVS and the indicated FLAG-TRAF3 constructs (wtTRAF3 or TRAF3-AKKFF). 24 h post-transfection, whole cell extracts were prepared using 1% Triton X-100 or RIPA lysis buffers as indicated. Cellular extracts were then subjected to immunoprecipitation using anti-FLAG antibodies or used in Western blot analysis (Input). Following multiples washing steps, immunoprecipitated proteins were then subjected to Western blot analysis using the indicated antibodies. Interestingly, using a soft lysis condition (1% Triton X-100), we were not able to extract the same amount of the two TRAF3 populations in the IP and in the INPUT (left panels), most likely due to the ability of the TRAF3-AKKFF mutant to be retained in the rich vesicular COPI/COPII environment. On the other hand, the use of a RIPA buffer helped the extraction of the TRAF3-AKKFF mutant from its vesicularrich environment (right panels). However under these conditions, we might have disrupted the COPI/COPII vesicles, releasing the TRAF3-AKKFF mutant into the cell lysate an