Biochemical Functions of the Membrane-Binding Domain of CARMIL.

Actin assembly at membranes is often associated with proteins with 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 capping protein binding region (CBR) of CARMIL includes a CPI motif and a CSI motif, followed by a membrane binding (MB) domain. The MB domain is necessary for the function of CARMIL in cells, and it is sufficient for targeting GFP to the plasma membrane of cells. 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, bringing the CPI and CSI motifs to the bead, and thus activating 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. We also report that the MB domain released from the membrane enhances the ability of the CPI and CSI domains to activate CP. 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.