Fig. 4. The CMS linker region plays a crucial role in maintaining the actin-binding activity of CMS. (A) The linker sequences of CMS and CIN85 were aligned using the BLAST program. Schematic representation of the C-terminal half of CMS and CIN85 depicting the region of identified similarities (boxed area). Sequence alignment of this region is shown below. Black bars represent the PR regions, gray bars represent the CC domain, solid lines mark the linker sequence. (B) Schematic representation of CMS CT in relation to the C-terminus of CIN85 (CIN85 CT), and a hybrid peptide of the C-termini of CMS and CIN85 (Hybrid), in which the linker sequence is replaced by the corresponding sequence of CIN85 (domains in CMS are depicted as in Fig. 3A; black rectangle in CIN85 CT represent sequences rich in prolines, striped rectangle represents linker sequence). Depicted GST-tagged peptides were purified, analyzed using Coomassie-stained acrylamide gels (left panel), and assayed for F-actin-binding activity in GST pull-down reactions. GST was included as a negative control. The reactions were subjected to western blot analysis to score for bound actin. Summary of relative values ± s.d. of actin-binding represents the average of four independent experiments (n=4). P values were calculated using Student's t-test. (C) The PR region of CIN85 contributes to actin-binding. Indicated GST-tagged peptides or GST alone were assessed for F-actin-binding in pull-down reactions. Reactions were analyzed as described.