K313 is contributing and K345 is essential to the plasma membrane recruitment of the TRAF domain.

<p>(A) Colocalization of the complete TRAF4 protein and of the TRAF domain in isolation with the TJ protein ZO-1 in MCF7 cells. Cells transiently transfected with YFP-tagged (green) complete TRAF4 protein (a) and TRAF domain (b) were labeled for endogenous ZO-1 (red) and DNA (nuclei in blue). Confocal sections are shown together with xz- and yz-scans. Schematic representation of the localization of the EYFP-tagged proteins is presented at the bottom. TRAF-EYFP exhibited recruitment all along the plasma membrane, whereas TRAF4-EYFP is specifically targeted to the TJs. (B) Colocalization between the TRAF4-TRAF domain and fluorescently tagged PIP-probes. MCF7 cells were co-transfected with the mCherry-tagged TRAF domain and EGFP-tagged PH domains of the phospholipase C protein (PH-PLC-GFP, top) and Akt (PH-Akt-GFP, bottom). Confocal sections showed that the TRAF domain of TRAF4 co-localized with PH-PLC-GFP and PH-Akt-GFP proteins that bind PI(4,5)P2 and PI(3,4,5)P3 at the apical and basolateral side of the cell, respectively. Nuclei were stained using Hoechst (blue). Insets on the right represent a 2.5× magnification. Scale bar, 5 µm. (C) Tubulation assays in COS7 cells. The recruitment of PH-PLC-Cherry (a), WT TRAF-Cherry (b), and mutant TRAF domains TRAF-K313E-Cherry (c) or TRAF-K345E-Cherry (d) on membrane tubes was studied by colocalization with BIN1/BAR-GFP (top panels) and BIN1/BAR-PI-GFP (bottom panels). Nuclei were stained using Hoechst (blue). Similarly to PH-PLC-Cherry, a known PI(4,5)P2 binding domain, TRAF-Cherry protein was specifically recruited to PIP-enriched membrane tubes. Both lysines 313 (c) and 345 (d) mutations prevent the recruitment of the TRAF domain to PIP-enriched membrane tubes. (a–d) Confocal sections; insets on the right are 3.5× magnification, Scale bar, 5 µm. (D) Confocal sections of MCF7 cells transfected with WT and mutant TRAF domains of TRAF4 fused to EYFP (green). The WT TRAF domain is recruited to the plasma membrane (top panels). While the K313E mutant (middle panel) is mostly cytoplasmic, a small fraction of the protein still localizes to the plasma membrane (arrows). The K345E mutant is only detected in the cytoplasm (bottom panel). Nuclei were stained using Hoechst (blue). Insets on the right are 3× magnification. Scale bar, 5 µm.