Native cyclase-associated protein and actin fromXenopus laevisoocytes form a 4:4 complex with a tripartite structure

Cyclase-associated protein (CAP) is a conserved actin-binding protein that regulates multiple aspects of actin filament dynamics, including polymerization, depolymerization, filament severing, and nucleotide exchange. Intriguingly, CAP has been isolated from different cells and tissues as an equimolar complex with actin, and previous studies have shown that a CAP-actin complex contains six molecules each of CAP and actin. Here, we successfully isolated a complex of Xenopus cyclase-associated protein 1 (XCAP1) and actin from oocyte extracts and demonstrated that the complex contained four molecules each of XCAP1 and actin. The XCAP1-actin complex remained stable as a single population of 340 kDa in hydrodynamic analysis using gel filtration or analytical ultracentrifugation. Examination of the XCAP1-actin complex by high-speed atomic force microscopy revealed a tripartite structure: a middle globular domain and two globular arms. The two arms were connected with the middle globular domain by a flexible linker and observed in two states with different heights, presumably representing the presence or absence of G-actin. We hypothesize that the middle globular domain corresponds to a tetramer of the N-terminal helical-folded domain of XCAP1, and that each arm in the high state corresponds to a hetero-tetramer containing a dimer of the C-terminal CARP domain of XCAP1 and two G-actin molecules. This novel configuration of a CAP-actin complex may represent a functionally important aspect of this complex.