CARMIL membrane-binding domain regulates capping protein and actin assembly

Actin assembly at membranes is associated with protein domains that bind and regulate heterodimeric actin capping protein (CP). CP-binding domains can target CP to the membrane and activate CP by promoting dissociation of its stoichiometric inhibitor V-1. The CP-binding region of CARMIL includes a CPI motif and a CSI motif, followed by a membrane binding (MB) domain. The MB domain is necessary for CARMIL function in cells, and it can target GFP to the plasma membrane. Here, we investigated the mechanism and significance of the relationship of the MB domain to CP activity, including capping of actin filament barbed ends and promotion of Arp2/3-nucleated actin assembly. We found that the MB domain is able to bind to lipid-coated beads, bring the CPI and CSI motifs to the bead, and thus activate CP to promote Arp2/3-based actin assembly. In addition, we discovered that the MB domain can dissociate from the lipid membrane once CP binds; this observation may help account for the long-standing quandary as to how activated CP is released from the membrane and how CP functions to activate Arp2/3-mediated actin assembly near the membrane. Thus, the CARMIL MB domain has multiple biochemical functions regulating actin assembly at a membrane. First, it can target CARMIL, CP, and barbed ends to the plasma membrane. Second, the MB domain can leave the membrane, and this promotes the uncapping of capped barbed ends and activates soluble CP, with greater ability than seen with the membrane-attached state.