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

Biochemistry. 2001 Sep 25;40(38):11390-404. doi: 10.1021/bi010486b.

Abstract

Actin-based propulsion of the bacteria Listeria and Shigella mimics the forward movement of the leading edge of motile cells. While Shigella harnesses the eukaryotic protein N-WASp to stimulate actin polymerization and filament branching through Arp2/3 complex, the Listeria surface protein ActA directly activates Arp2/3 complex by an unknown mechanism. Here we show that the N-terminal domain of ActA binds one actin monomer, in a profilin-like fashion, and Arp2/3 complex and mimics the C-terminal domain of WASp family proteins in catalyzing filament barbed end branching by Arp2/3 complex. No evidence is found for side branching of filaments by ActA-activated Arp2/3 complex. Mutations in the conserved acidic (41)DEWEEE(46) and basic (146)KKRRK(150) regions of ActA affect Arp2/3 binding but not G-actin binding. The motility properties of wild-type and mutated Listeria strains in living cells and in the medium reconstituted from pure proteins confirm the conclusions of biochemical experiments. Filament branching is followed by rapid debranching. Debranching is 3-4-fold faster when Arp2/3 is activated by ActA than by the C-terminal domain of N-WASp. VASP is required for efficient propulsion of ActA-coated beads in the reconstituted motility medium, but it does not affect the rates of barbed end branching/debranching by ActA-activated Arp2/3 nor the capping of filaments. VASP therefore affects another still unidentified biochemical reaction that plays an important role in actin-based movement.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actin-Related Protein 2
  • Actin-Related Protein 3
  • Actins / metabolism*
  • Amino Acid Sequence
  • Animals
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Base Sequence
  • Conserved Sequence
  • Cytoskeletal Proteins*
  • DNA Primers
  • Humans
  • Kinetics
  • Listeria monocytogenes / genetics
  • Listeria monocytogenes / physiology*
  • Membrane Proteins / chemistry
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Microfilament Proteins / chemistry
  • Microfilament Proteins / metabolism
  • Models, Chemical
  • Molecular Sequence Data
  • Movement
  • Muscle, Skeletal / metabolism
  • Mutagenesis, Site-Directed
  • Nerve Tissue Proteins / chemistry*
  • Nerve Tissue Proteins / metabolism
  • Polymerase Chain Reaction
  • Rabbits
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Shigella / genetics
  • Shigella / physiology
  • Wiskott-Aldrich Syndrome Protein, Neuronal

Substances

  • ACTR2 protein, human
  • ACTR3 protein, human
  • Actin-Related Protein 2
  • Actin-Related Protein 3
  • Actins
  • Bacterial Proteins
  • Cytoskeletal Proteins
  • DNA Primers
  • Membrane Proteins
  • Microfilament Proteins
  • Nerve Tissue Proteins
  • Recombinant Proteins
  • WASL protein, human
  • Wiskott-Aldrich Syndrome Protein, Neuronal
  • actA protein, Listeria monocytogenes