Figure 1: TBC1d5 forms a tight complex with the retromer CSC.
From: Structural and mechanistic insights into regulation of the retromer coat by TBC1d5
(a) Schematic representation of retromer CSC. (b) GST–TBC1d5 pull down of purified retromer CSC. Shown is the TBC1d5 constructs used (top) and a Coomassie blue stained SDS–PAGE gel of bound samples. Open triangles indicate GST–TBC1d5 or GST proteins. (c) ITC of TBC1d5TBC titrated into CSC in buffer containing 20 mM Tris-HCl pH 8.0, 200 mM NaCl and 5 mM βME at 20 °C. Top and bottom panels show raw and integrated heat from injections, respectively. The black curve in the bottom panel represents a fit of the integrated data to a single-site binding model. (d) Affinity between CSC and TBC1d5TBC WT, ΔIns1 or ΔIns2 determined by ITC. Association constant (Ka) are shown together with errors from data fitting. (e) Subcellular localization of TBC1d5 WT, ΔIns1 and ΔIns2. Hela cells were transfected with yellow fluorescent protein (YFP) or various YFP–TBC1d5 (green) and then fixed and labelled with anti-VPS35 antibodies (red). The larger images from which these were captured are shown in Supplementary Fig. 3B. Scale bar, 5 μm.
