Smaug and SMO physically interact and colocalize (A) Coimmunoprecipitation of SMO-HA and Myc-Smaug proteins.Figure 1(A) Coimmunoprecipitation of SMO-HA and Myc-Smaug proteins. Extracts of Cl8 cells expressing combinations of SMOWT-HA or SMOPKA-SD-HA with Myc-SmaugWT in the absence or presence of HH were immunoprecipitated (IP) with anti-HA. The input (lower panel) and the IP complexes (upper panel) were analyzed by western blot with anti-Myc (αMyc) or anti-HA (αHA) antibodies. Here and in the other figures, the names of the proteins detected are indicated on the left and the molecular weights on the right, in kDa; the samples loaded for the input and the supernatants are equivalent to a tenth of the volume loaded for the IPs. * corresponds to higher molecular weight forms of SMO of unknown origin. Here and in panel F, the black arrow indicates unphosphorylated SMOWT-HA and the bracket indicates the phosphorylated forms of SMOWT-HA or SMOPKA-SD. (B) Schematic representation of the domain structure of the Smaug protein. The conserved SSR 1 and 2 are shown in blue. They are separated by a 79 amino-acid-long non-conserved region (here referred to as 'M'). The sterile alpha motif (SAM) domain is in green and the pseudo heat analogous topology domain (PHAT domain, which increases the affinity of the SAM domain for SRE) in yellow. The dashed double-arrow line at the top represents the smallest interacting region (called SID for Smallest Interacting Domain) found according the two-hybrid screen. The truncated constructs used are presented below. The ability to interact with SMOWT-HA is indicated on the right: in green for yes, red for no. The full red double-arrow line below represents the smallest SMO-binding region (BR) region that we could identify. The amino acid numbers correspond to Smaug-PA. (http://flybase.org/reports/FBgn0016070.html). See also Fig EV1A-C. (C) Schematic representation of the C-terminal 'cytotail' of SMO. The PKA/CKI and FU phosphorylated regions are indicated as orange and green boxes, respectively. The FU-binding region (BR) is indicated by a full green double arrow above. The truncated constructs used are presented below and their ability to coIP with Myc-SmaugWT is indicated on the right. The full red double-arrow line at the bottom represents the smallest Smaug binding region (BR) region identified in this work. (D, E) Mapping of the Smaug interaction domain in SMO. Extracts from Cl8 cells transfected with Myc-SmaugWT and various regions of SMO fused to either HA (D) or to GFP (E) were immunoprecipitated with αMyc (D) or αGFP (E) and analyzed by western blotting with αMyc (lower panel in D and upper panel in E), αHA (D, upper panel) or αGFP (E, lower panel). In: Input; Sup: supernatant. See also Fig EV1D-F. (F) Hyperphosphorylated forms of SMO do not interact with Smaug. Left panel: Extracts of Cl8 cells expressing SMOWT-HA with or without Myc-SmaugWT, in the absence or presence of HH were IP with an αMyc antibody before analysis by western blot with αHA (upper panel) or αMyc antibodies (lower panel). Right panel: Cl8 cells expressing SNAP-SMOWT with Myc-SmaugWT in the presence of HH were extracellularly labelled with a membrane-nonpermeable fluorescent SNAP substrate before cell lysis and immunoprecipitation (IP) with anti-Myc. Labelled SNAP-SMO was directly visualized after electrophoresis. Note its presence in the IP fraction with a relative enrichment of the less phosphorylated forms compared to the hyperphosphorylated forms. See also Fig EV1G-J. (G) SMO and Smaug colocalize. Representative fluorescent images of Cl8 cells expressing GFP-SmaugWT alone (1-2) or together with SMOWT-mCherry (3-3'' and 4-4'') either without (1, 3-3") or with HH (2, 4-4"). The merge images in 3" and 4" show GFP-SmaugWT in green and SMOWT-mCh in red. The same results were seen with different fluorescent tags as well (Fig EV2B). The lack of effect of HH on GFP-SmaugWT in absence of SMOWT-mCh is likely due to limiting amounts of endogeneous SMO. A least 20 cells were assayed for each condition. In absence of HH, all co-transfected cells exhibited greater than 90% colabelled SMOWT-mCherry. Scale bar (shown in G1, identical for all panels):10μm. See also Fig EV2.