Listeria protein ActA mimics WASP family proteins: It activates filament barbed end branching by Arp2/3 complex

Actin-based propulsion of the bacteria Listeria and Shigella mimics the for ward movement of the leading edge of motile cells. While Shigella harnesses the eukaryotic protein N-WASp to stimulate actin polymerization and filame nt branching through Arp2/3 complex, the Listeria surface protein ActA dire ctly activates Arp2/3 complex by an unknown mechanism. Here we show that th e N-terminal domain of ActA binds one actin monomer, in a profilin-like fas hion, and Arp2/3 complex and mimics the C-terminal domain of WASp family pr oteins in catalyzing filament barbed end branching by Arp2/3 complex. No ev idence is found for side branching of filaments by ActA-activated Arp2/3 co mplex. Mutations in the conserved acidic (DEWEEE46)-D-41 and basic (KKRRK15 0)-K-146 regions of ActA affect Arp2/3 binding but not G-actin binding. The motility properties of wild-type and mutated Listeria strains in living ce lls and in the medium reconstituted from pure proteins confirm the conclusi ons of biochemical experiments. Filament branching is followed by rapid deb ranching. Debranching is 3-4-fold faster when Arp2/3 is activated by ActA t han by the C-terminal domain of N-WASp. VASP is required for efficient prop ulsion of ActA-coated beads in the reconstituted motility medium, but it do es not affect the rates of barbed end branching/ debranching by ActA-activa ted Arp2/3 nor the capping of filaments. VASP therefore affects another sti ll unidentified biochemical reaction that plays an important role in actin- based movement.