Figure 5

Copolymerisation of cofilin–actin fusion protein with K336I actin affects cleavage of the D-loop in the fusion protein. As shown in our previous report³¹, the fusion protein polymerises and depolymerises normally in a salt-dependent manner, and the cofilin moiety in the fusion protein has a pH-sensitive depolymerisation and severing activities of actin filaments. These functional properties of the cofilin moiety are similar to that of normal cofilin. Thus, there is seems to be no steric problem between the actin and cofilin moieties in the fusion protein in terms of interaction with other actin molecules. (A) Schematic drawing showing the detection of structural changes of the actin moiety in the fusion protein when copolymerised with K336I actin. In a co-filament, when K336I actin protomers affect the structure of the WT actin moiety of the fusion protein, they reduce the affinity of that actin moiety for cofilin moiety, causing its D-loop to have reduced susceptibility to subtilisin cleavage. The D-loop of actin (except for the actin moiety in the fusion protein) is not shown. (B) Subtilisin digestions of K336I/cofilin–actin co-filaments or WT/cofilin–actin co-filaments. Cofilin–actin fusion protein with an intact D-loop became slightly smaller after treatment with subtilisin, presumably due to nicking in the cofilin moiety by subtilisin, as shown in our previous report³¹. Digestion of the D-loop in the cofilin–actin fusion protein yielded bands of approximately 30 and 36 kDa, and did not yield a 42 kDa band³¹. The amount of undigested D-loop of the fusion protein (the sum of the 66 kDa band and the slightly smaller nicked band, marked by asterisks) in the K336I/cofilin–actin co-filaments was greater than in the WT/cofilin–actin co-filaments when digested for the same length of time. Nicking of the cofilin moiety in the fusion protein was accelerated by copolymerisation with K336I actin, and was most evident after 11 minutes of subtilisin digestion. This is consistent with the results of co-sedimentation experiments (Fig. 4), and supports the model shown in (A). Full-length gel image is shown in Supplementary Fig. S8.