Reconstitution of human Arp2/3 complex reveals critical roles of individual subunits in complex structure and activity

The Arp2/3 complex is a seven-protein assembly that is critical for actin n ucleation and branching in cells. Here we report the reconstitution of acti ve human Arp2/3 complex after expression of all seven subunits in insect ce lls. Expression of partial complexes revealed that a heterodimer of the p34 and p20 subunits constitutes a critical structural core of the complex, wh ereas the remaining subunits are peripherally located. Arp3 is crucial for nucleation, consistent with it being a structural component of the nucleati on site. p41, p21, and p16 contribute differently to nucleation and stimula tion by ActA and WASP, whereas p34/p20 bind actin filaments and likely func tion in actin branching. This study reveals that the nucleating and organiz ing functions of Arp2/3 complex subunits are separable, indicating that the se activities may be differentially regulated in cells.