TJ recruitment of TRAF4 is PIP-binding dependent.

<p>(A) Schematic representation of sh-insensitive Flag-tagged WT and mutant TRAF4 constructs used to reintroduce TRAF4 expression in MCF7/shT4 cells. (B) Recruitment of WT and mutant TRAF4 proteins (green) at TJs was analyzed by colocalization with ZO-1 (red). The highlighted overlap (white) between TRAF4 and ZO-1 staining is shown on merge panels and alone on the right panel. While the TRAF4-K313E mutant is still partially colocalized with ZO-1 (middle panels), the TRAF4-K345E mutant does not colocalize anymore with ZO-1 (bottom panels). Scale bar, 10 µm. (C) Quantification of WT and mutant TRAF4 recruitment at TJs. The colocalization index (overlapping area between TRAF4 and ZO-1 staining divided by the TJ length) was measured on 10 microscopic fields. Compared to the WT protein, the colocalization index was reduced by 40% and 78% for K313E and K345E TRAF4 mutants, respectively. (D) Western blot analysis of TRAF4 protein level in parental MCF10A and in TRAF4-silenced cells (MCF10A/shT4) where WT (MCF10A/shT4+TRAF4) and mutant (MCF10A/shT4+TRAF4K345E) TRAF4 expression was restored. The MCF10A/shT4+pBABE cell line represents a control line transduced with the empty vector. Beta-actin was used as a loading control. (E) The presence of TJs was estimated by ZO-1 staining in parental (a) and in TRAF4-silenced cells where the expression of WT (c) and mutant TRAF4 (d) was reintroduced. TRAF4-silenced cell line transduced with the empty vector (b) was used as a control. The PIP-binding–deficient TRAF4-K345E cannot rescue the phenotype induced by TRAF4 silencing on TJs. Left panels, representative confocal image sections of ZO-1 staining (green); right panels, merge with Hoechst staining (blue). Scale bar, 20 µm. (F) TJ quantification in cell lines described in (D) and (E) was performed as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001726#pbio-1001726-g001" target="_blank">Figure 1E</a>. n, number of microscopic fields used for the quantification.</p

TRAF4 protein level modulates TJs in confluent MCF10A monolayers.

<p>(A) Western-blot analysis of TRAF4 in parental and in established MCF10A cell lines. To knock down TRAF4, parental MCF10A cells (lane 1) were transduced with a shRNA targeting TRAF4 (MCF10A/shT4, lanes 3–5); a nonspecific shRNA was used as control (MCF10A/shCtrl, lane 2). To restore TRAF4 expression, MCF10A/shT4 cells were transduced with a shT4-insensitive vector encoding TRAF4 (MCF10A/shT4+TRAF4, lane 5); the control cell line (MCF10A/shT4+pBABE, lane 4) was transduced with the empty vector. A gain of function and a control cell line were generated with a TRAF4 expression plasmid (MCF10A/TRAF4, lane 7) or the empty vector (MCF10A/pBABE, lane 6), respectively. TRAF4 expression levels normalized to actin are indicated. (B–D) The presence of TJs was estimated by ZO-1 staining in the different cell lines of TRAF4 loss of function (B), gain of function (C), and rescue (D) experiments. Left panels are representative confocal sections of ZO-1 staining (green), and right panels are merges with Hoechst staining (blue). Scale bar, 20 µm. (E) TJ quantification. Score representing the number of cells with a continuous ZO-1 staining, normalized to parental MCF10A cells (percentage). The number of microscopic fields used for the quantification is indicated at the bottom of the bar chart. TRAF4 knock-down increased the number of cells with TJs, whereas TRAF4 overexpression had the opposite effect.</p

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.</p

TRAF4 promotes MCF7 cell migration.

<p>(A) Western blot analysis of TRAF4 expression. In MCF7 cells, TRAF4 expression has been silenced (lanes 2–6), increased (lane 8), and restored in silenced cells using the WT (lane 5) and the K345E mutant (lane 6). Parental (lane 1), control shRNA (lane 2), and control expression vector (lane 7) together with a TRAF4 silenced line transduced with the empty vector (lane 4) were used as controls. TRAF4 expression levels were normalized to control parental cells using β-actin as loading control; values are indicated on the top. (B) Representative microscopic field of the bottom side of the transwell. Migrating cell nuclei were stained with Hoechst, and images are shown as inverted look-up table. (C) Bar chart representing the quantification of cell migration in MCF7 cells. The number of cells that migrated were counted and normalized to control parental cells. Thirty-six microscopic fields from three independent experiments were used for the quantification.</p

TRAF4 binds PIPs through its TRAF domain.

<p>(A) Schematic representation of the recombinant proteins used in lipid binding assays. TAP and 6His tags were used for the purification. RING, Zf, and TRAF are conserved structural domains present in the TRAF4 protein. (B) Coomassie blue staining (a) and Western blot analysis (b) of purified recombinant proteins. The antibody used recognized the immunoglobulin-binding domain of protein A from the TAP tag. TRAF4 degradation products are indicated by asterisks. (C) Lipid-overlay assay. Left, schematic view of a PIP-strip membrane. LPA, lysophosphatidic acid; S1P, Sphingosine-1-phosphate; LPC, Lysophosphocholine; PI, Phosphatidylinositol; PI(3)P, PI-(3)-phosphate; PI(4)P, PI-(4)-phosphate; PI(5)P, PI-(5)-phosphate; PI(3,4)P2, PI-(3,4-)bisphosphate; PI(3,5)P2, PI-(3,5)-bisphosphate; PI(4,5)P2, PI-(4,5)-bisphosphate; PIP(3,4,5)P3, PI-(3,4,5)-trisphosphate; PA, Phosphatidic acid; PE, Phosphatidylethanolamine; PS, Phosphatidylserine; PC, Phosphatidylcholine. The TAP-6His recombinant protein served as a negative control. Immunodetection of bound proteins was performed using a TAP-identifying antibody. TAP-6His and RING-7xZf did not bind to any membrane-coated lipids, while both full-length TRAF4 and the TRAF domain in isolation interacted with all PIPs and PA. (D) The TRAF domain of TRAF4 binds PIP in solution. Electrospray ionization time-of-flight mass spectrometry deconvoluted spectra of the TRAF domain of TRAF4 in the absence (a) and in the presence (b) of PI(3,4,5)P3-diC4. In isolation and in the absence of lipid, the TRAF domain is a trimer (a). In the presence of PIP, three additional peaks corresponding to one to three bound lipids are detected (b). Theoretical masses of the TRAF trimer and PI(3,4,5)P3-diC4 are 67.678 kDa and 0.714 kDa, respectively. (E) Liposomes flotation assay. a, schematic representation of the liposome flotation assay. Blank liposomes, PI(4,5)P2-containing liposomes, and PI(3,4,5)P3-containing liposomes were incubated with recombinant proteins, and liposome/protein-mixed fractions were separated by sucrose gradient ultracentrifugation. Binding of recombinant control TAP-6HIS (b) and TRAF domain of TRAF4 (c) to liposomes using membrane flotation assay. Fluorescent analyses (dot blot) of NBD-PE indicated that blank and PIP-containing liposomes were present in the top fraction. The presence of recombinant proteins in each fraction was detected by Western blot using anti-His antibody and quantified by densitometry-analysis using ImageJ software. The control TAP-6His was predominantly detected in the bottom fraction (b). In contrast the centrifugation profile of the TRAF4-TRAF domain was modified in the presence of PIP-containing liposomes (c). Indeed when mixed with blank liposomes, the TRAF domain was present in the bottom fraction, while in the presence of PI(4,5)P2) and PI(3,4,5)P3-containing liposomes, the TRAF domain was present in the top fraction. (F) Affinity of TRAF4 for PIP was measured by ITC. Titration was performed with 16 µM TRAF-6His recombinant protein, to which 500 µM of inositol-(1,3,4,5)-tetrakisphosphate were added incrementally. The TRAF domain of TRAF4 binds IP4 with a K_D of 5.68 µM.</p

Modeling of the PIP3-diC4 binding onto the TRAF domain of TRAF4.

<p>(A) Model of PIP3-diC4 binding onto the TRAF domain of TRAF4 in ribbon drawing (a) and surface (b) representations. The PIP3-diC4 is bound in a pocket at the interface between two different TRAF monomers. (B) PIP3-diC4-interacting residues and PIP3-diC4 ligand are depicted in stick models. Phosphate, nitrogen, and oxygen atoms are colored in orange, blue, and red, respectively. Hydrogen bond interactions are shown as dashed lines. In this model, three basic residues (R297, K313, and K345) and one aromatic residue (Y338) interact directly with the lipid.</p

The PIP-binding ability is conserved through the TRAF family.

<p>(A) Coomassie blue staining (a) and Western blot analysis (b) of purified recombinant TRAF domains from the TRAF family. The antibody used recognized the immunoglobulin-binding domain of protein A from the TAP tag. (B) Lipid-overlay assay of TRAF domains from the TRAF family. Left, schematic view of a simplified PIP-strip. In this assay, the TAP-6His and the TRAF of TRAF4 are used as negative and positive control, respectively. Immunodetection of membrane-bound proteins was performed as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001726#pbio-1001726-g002" target="_blank">Figure 2C</a>. All TRAF domains from the TRAF family bind to PIPs. (C) (a) Binding of recombinant TRAF domains of TRAF4, TRAF5, and TRAF6 to liposomes was analyzed using liposome flotation assay as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001726#pbio-1001726-g002" target="_blank">Figure 2E</a>. The control TAP-6His is unable to float in the presence of control and PIP-containing liposomes. In contrast, recombinant TRAF domains of TRAF5 and TRAF6 floated specifically when bound to PIP-enriched liposomes. (b) The quantification of proteins present in the different fractions was performed by Western blot and densitometry using ImageJ software.</p

Structural alignment of CRTI with five FAD-binding Rossmann fold proteins (Pfam:CL0063) identified by a DALI search.

<p> The proteins are <i>Methanosarcina mazei</i> oxidoreductase (RMSD 4.6; 3 KA7; Seetharaman <i>et al</i>., unpublished), <i>Myxococcus xanthus</i> protoporphyrinogen oxidase (RMSD 4.6; 2 IVD; Corradi <i>et al</i>., 2006), <i>Nicotiana tabacum</i> mitochondrial protoporphyrinogen IX oxidase (RMSD 4.8; ISEZ; Koch <i>et al</i>., 2004), <i>Bacillus subtilis</i> protoporphyrinogen oxidase (RMSD 5.3, 3I6D, Qin <i>et al</i>., 2010) and <i>Rhodococcus opacus</i> L-amino acid oxidase (RMSD 4.8; 2JB2; Faust <i>et </i><i>al</i>., 2007). The secondary structure elements of CRTI have been indicated above the alignment and the colored bar underneath the alignment indicates the domain organisation with the FAD-binding domain (green), the substrate-binding domain (blue), and the non-conserved ‘helical’ or ‘membrane-binding’ domain (orange). Disordered regions in the structure are represented by a dotted line and putative FAD binding residues are indicated by purple circles (hydrophobic interactions) and triangles (hydrophilic interactions). This figure was generated with TEXshade <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039550#pone.0039550-Beitz1" target="_blank">[60]</a>.</p

Electron transfer reactions catalyzed by CRTI.

<p>A, Potentiometric measurement of oxygen consumption during phytoene desaturation. B, Phytoene desaturation (lycopene formation) using quinones as electron acceptors. The assays were run under an N₂ atmosphere for 30 minutes otherwise maintaining the standard conditions. The quinones used were menaquinone (−80 mV), phylloquinone (−70 mV), menadione (0 mV), duroquinone (+5 mV), Q10 (+65 mV), naphtoquinone (+70 mV) dichlophenolindophenol (+217 mV) and benzoquinone (+280 mV) all at a concentration of 240 µM. Open squares, naphtoquinones, filled symbols, benzoquinones.</p

Phytoene desaturation – “complex” vs. “simple”.

<p>Left, the plant/cyanobacterial system consisting of the two desaturases, phytoene desaturase (PDS) and ζ-carotene desaturase (ZDS). The pathway involves specific poly-<i>cis</i>-intermediates and results in the formation of 7,9,9′7′-tetra-<i>cis</i>-lycopene ( =  prolycopene). <i>Cis</i>-<i>trans</i> isomerases act at the 9,15,9′-tri-<i>cis</i>-ζ-carotene (Z-ISO) and prolycopene (CRTISO) stage, the latter forming all-<i>trans</i>-lycopene, the substrate for lycopene cyclases. The electron acceptors identified so far for PDS (assumed here to be the same for the related ZDS) are plastoquinone and the plastoquinone:oxygen oxidoreductase PTOX. The necessity for an electron donating branch, resulting in redox chains into which PDS integrates has been suggested. Right, CRTI-mediated phytoene desaturation encompassing all four desaturation steps and one <i>cis</i>-<i>trans</i> isomerization step to form all-<i>trans-</i>lycopene. The desaturase CRTI and the isomerase CRTISO share sequential similarity.</p