The RhoGEF TEM4 Contains an F-Actin Binding Domain Figure 4. TEM4 binds directly to F-actin with high affinity. A, Binding of TEM4 (residues 81–135) to F-actin was examined by actin co- sedimentation assays. Recombinant GST-TEM4 protein was incubated with F-actin and subjected to high speed centrifugation. Soluble (s) and pellet (p) fractions were resolved by SDS-PAGE and stained with Coomassie Blue. B, Coomassie Blue-stained gel showing the near-saturation binding of GST-TEM4-ABD to F-actin. Numbers below protein bands indicate protein concentrations in mM as determined by densitometry. C, TEM4 ABD binds F-actin. Silver stained gel shows co-sedimentation of untagged TEM4 ABD with F-actin. D-G, TEM4 preferentially binds dynamic ATP/ADP-Pi-F-actin. ATP- or ADP-bound G-actin was polymerized and binding of the resulting F-actin filaments to GST-ABD was determined by co-sedimentation analysis. Representative Coomassie Blue-stained gels showing amount of ATP/ADP-Pi- or ADP-F-actin in the pellet of co-sedimentation experiment (D and E, top panel). Immunoblot showing amount of GST-ABD protein left in the supernatant in co-sedimentation experiments (bottom panel). F, G, Equilibrium binding of GST-ABD to ATP/ADP-Pi- or ADP-F-actin. Amount of GST-ABD bound to F-actin was calculated from immunoblots similar to the ones shown in D and E. doi:10.1371/journal.pone.0041876.g004 To identify the minimal domain required for the association with F-actin in vivo, we performed extensive deletion mutagenesis and identified a region encompassing residues 80–135 that exhibited cellular localization similar to TEM4 1–582 (Fig. 3A). The 81–135 region of human TEM4 is highly conserved within TEM4 orthologs (Fig. 3B), but does not exhibit sequence similarity to known actin-binding domains [29–31]. Therefore, TEM4 81– 135 is sufficient for localization to the actin cytoskeleton in vivo and may constitute a novel actin binding domain (ABD). TEM4 Binds F-actin Through the Novel Actin-Binding Domain To determine if the association of TEM4 with F-actin is direct, we purified recombinant GST-TEM4 81–135 (GST-ABD) and evaluated its ability to bind purified F-actin using a high-speed actin co-sedimentation assay. Recombinant GST-ABD was found predominantly in the F-actin-containing pellet (p) only in the presence of F-actin (Fig. 4A). The stoichiometry of GST-ABD binding to F-actin was estimated to be 1:1.4 (Fig. 4B). However, when we accounted for the 15–20% GST cleavage product present in the GST-ABD protein sample, the stoichiometry came close to a 1:1 ratio. The specificity of interaction between TEM4 ABD and F-actin was further confirmed by co-sedimentation of F- actin with the untagged TEM4 ABD (Fig. 4C). These data demonstrate that sequences within TEM4 81–135 comprise a high- affinity ABD. In a cell, newly polymerized, dynamic actin filaments are composed mainly of ATP- and ADP-Pi–actin [32], while mature filaments are composed primarily of ADP-actin [33,34]. The PLoS ONE | www.plosone.org 6 July 2012 | Volume 7 | Issue 7 | e41876