Abstract
The Arp2/3 (actin-related protein 2/3) complex nucleates branched actin filaments involved in multiple cellular functions, including endocytosis and cellular motility. Two subunits (Arp2 and Arp3) in this seven-subunit assembly are closely related to actin and upon activation of the complex form a "cryptic dimer" that stably mimics an actin dimer to nucleate a new filament. Both Arps contain a shared actin core structure, and each Arp contains multiple insertions of unknown function at conserved positions within the core. Here we characterize three key insertions within the actin core of Arp3 and show that each one plays a distinct role in modulating Arp2/3 function. The β4/β5 insert mediates interactions of Arp2/3 complex with actin filaments and "dampers" the nucleation activity of the complex. The Arp3 hydrophobic plug plays an important role in maintaining the integrity of the complex but is not absolutely required for formation of the daughter filament nucleus. Deletion of the αK/β15 insert did not constitutively activate the complex, as previously hypothesized. Instead, it abolished in vitro nucleation activity and caused defects in endocytic actin patch assembly in fission yeast, indicating a role for the αK/β15 insert in the activated state of the complex. Biochemical characterization of each mutant revealed steps in the nucleation pathway influenced by each Arp3-specific insert to provide new insights into the structural basis of activation of the complex.
Highlights
The Arp2/3 complex is a molecular machine that nucleates branched actin filaments using poorly understood structural mechanisms
The Arp3 4/5 Insert Mediates Interactions of Arp2/3 Complex with Actin Filaments—The loop between 4 and 5 is a hotspot for insertions within the actin core [23], and in actin this loop, called the DNaseI loop, makes intersubunit contacts within filaments [37,38,39]
In crystal structures of Arp2/3 complex, the 4/5 loop is disordered [4, 5], but the EM model of a branch junction places it near the mother filament of actin [6], and molecular dynamics simulations indicate that it forms favorable contacts with the filament [40] (Fig. 1, A and B)
Summary
The Arp2/3 complex is a molecular machine that nucleates branched actin filaments using poorly understood structural mechanisms. The Arp2/3 (actin-related protein 2/3) complex nucleates branched actin filaments involved in multiple cellular functions, including endocytosis and cellular motility. Two subunits (Arp and Arp3) in this seven-subunit assembly are closely related to actin and upon activation of the complex form a “cryptic dimer” that stably mimics an actin dimer to nucleate a new filament. Both Arps contain a shared actin core structure, and each Arp contains multiple insertions of unknown function at conserved positions within the core. Residues at the barbed end of Arp contact the actin subunit at the end of the daughter
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