title: A novel actin-binding motif in Las17/WASP nucleates actin filaments independently of Arp2/3.
abstract: BACKGROUND
  Actin nucleation is the key rate-limiting step in actin polymerization, and tight regulation of this process is critical to ensure that actin filaments form only at specific regions of the cell. Las17 is the primary activator of Arp2/3-driven actin nucleation in yeast and is required for membrane invagination during endocytosis. Its mammalian homolog, WASP, has also been studied extensively as an activator of Arp2/3-driven actin polymerization. In both Las17 and WASP, actin nucleation activity is attributed to an ability to bind actin through a WH2 domain and to bind Arp2/3 through an acidic region. The central region of both Las17 and WASP is rich in proline residues and is generally considered to bind to SH3-domain-containing proteins.
  
  
  RESULTS
  We have identified a novel actin-binding activity in the polyproline domain of both yeast Las17 and mammalian WASP. The polyproline domain of Las17 is also able to nucleate actin filaments independently of Arp2/3. Mutational analysis reveals that proline residues are required for this nucleation activity and that the binding site on actin maps to a region distinct from those used by other nucleation activities. In vivo analysis of yeast strains expressing las17 mutated in the WH2 domain, one of its proline motifs, or both shows additive defects in actin organization and endocytosis, with the proline mutant conferring more severe phenotypes than the WH2 mutant.
  
  
  CONCLUSIONS
  Our data demonstrate a new actin-binding and nucleation mechanism in Las17/WASP that is required for its function in actin regulation during endocytosis.
authors.affiliation: Department of Biomedical Science, Firth Court, University of Sheffield, Sheffield S10 2TN, UK.
authors.forename: Agnieszka N
authors.initials: AN
authors.lastname: Urbanek
authors.forename: Adam P
authors.initials: AP
authors.lastname: Smith
authors.forename: Ellen G
authors.initials: EG
authors.lastname: Allwood
authors.forename: Wesley I
authors.initials: WI
authors.lastname: Booth
authors.forename: Kathryn R
authors.initials: KR
authors.lastname: Ayscough
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D051376:Actin-Related Protein 2-3 Complex / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D004705:Endocytosis; D058105:Polymerization; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D051316:Wiskott-Aldrich Syndrome Protein / Q000378:metabolism*
chemical_list: D051376:Actin-Related Protein 2-3 Complex; D000199:Actins; C480361:LAS17 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D051316:Wiskott-Aldrich Syndrome Protein