Fig. 4. The N-terminal domain of CAP1 is responsible for its cofilin-dependent interaction with unpolymerized actin. (A) The cofilin-actin complex interacts with CAP1-NT but not with CAP1-CT. G-actin (29 µM) was converted to the Mg-ATP-bound form by a 5 minute incubation in 40 µM MgCl₂ plus 0.5 mM EGTA. 15 µM of each His₆-tagged CAP1 domain was mixed with 7.2 µM of G-actin, 15 µM cofilin, or both, in a physiological salt buffer (lanes 1-6) or in a low salt buffer (lanes 7-12) at 2°C. Then, His₆-tagged proteins and associated molecule(s) were adsorbed to a Ni²⁺-resin. The resin was washed four times with 10 mM Tris-HCl, 1 mM MgCl₂, 50 mM KCl, 0.01% Triton X-100, 0.1 mM ATP, 0.1 mM DTT (pH 7.5), then boiled in SDS sample buffer. The bound and unbound fractions were analyzed by SDS-PAGE. NT-His designates the His₆-tagged N-terminal domain (amino acids 1-229) of CAP1, and HSE-CT designates the C-terminal domain (amino acids 255-475) carrying both His₆- and S-tags. (B,C) Co-sedimentation of the CAP1 domains with F-actin. F-actin (7.2 µM for B and 6 µM for C) was reacted with various amounts of CAP1-CT (B) or CAP1-NT (C) in the presence (+ cof) or absence (- cof) of cofilin (4 µM cofilin for B and 6 µM for C). After 6 hours, the samples were centrifuged at 190,000 g for 20 minutes with a Beckman TLA100 rotor. The supernatant and precipitate were subjected to SDS-PAGE. The amount of sedimented protein was quantified with a densitometer and plotted as a function of the amount of the respective CAP1 domains. Both of the CAP domains did not sediment in the absence of F-actin (data not shown).