ABSTRACT
Actin-related protein (Arp) 2/3 complex mediates the formation of actin filament branches during endocytosis and at the leading edge of motile cells. The pathway of branch formation is ambiguous owing to uncertainty regarding the stoichiometry and location of VCA binding sites on Arp2/3 complex. Isothermal titration calorimetry showed that the CA motif from the C terminus of fission yeast WASP (Wsp1p) bound to fission yeast and bovine Arp2/3 complex with a stoichiometry of 2 to 1 and very different affinities for the two sites (K(d)s of 0.13 and 1.6 µM for fission yeast Arp2/3 complex). Equilibrium binding, kinetic, and cross-linking experiments showed that (i) CA at high-affinity site 1 inhibited Arp2/3 complex binding to actin filaments, (ii) low-affinity site 2 had a higher affinity for CA when Arp2/3 complex was bound to actin filaments, and (iii) Arp2/3 complex had a much higher affinity for free CA than VCA cross-linked to an actin monomer. Crystal structures showed the C terminus of CA bound to the low-affinity site 2 on Arp3 of bovine Arp2/3 complex. The C helix is likely to bind to the barbed end groove of Arp3 in a position for VCA to deliver the first actin subunit to the daughter filament.
Subject(s)
Actin-Related Protein 2/metabolism , Actin-Related Protein 3/metabolism , Cattle/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , Wiskott-Aldrich Syndrome Protein/metabolism , Actin-Related Protein 2/chemistry , Actin-Related Protein 3/chemistry , Animals , Binding Sites , Crystallography, X-Ray , Polymerization , Protein Interaction Domains and Motifs , Protein Structure, Quaternary , Schizosaccharomyces/chemistry , Schizosaccharomyces pombe Proteins/chemistry , Thermodynamics , Wiskott-Aldrich Syndrome Protein/chemistryABSTRACT
Expression of human profilin-I does not complement the temperature-sensitive cdc3-124 mutation of the single profilin gene in fission yeast Schizosaccharomyces pombe, resulting in death from cytokinesis defects. Human profilin-I and S. pombe profilin have similar affinities for actin monomers, the FH1 domain of fission yeast formin Cdc12p and poly-L-proline (Lu, J., and Pollard, T. D. (2001) Mol. Biol. Cell 12, 1161-1175), but human profilin-I does not stimulate actin filament elongation by formin Cdc12p like S. pombe profilin. Two crystal structures of S. pombe profilin and homology models of S. pombe profilin bound to actin show how the two profilins bind to identical surfaces on animal and yeast actins even though 75% of the residues on the profilin side of the interaction differ in the two profilins. Overexpression of human profilin-I in fission yeast expressing native profilin also causes cytokinesis defects incompatible with viability. Human profilin-I with the R88E mutation has no detectable affinity for actin and does not have this dominant overexpression phenotype. The Y6D mutation reduces the affinity of human profilin-I for poly-l-proline by 1000-fold, but overexpression of Y6D profilin in fission yeast is lethal. The most likely hypotheses to explain the incompatibility of human profilin-I with Cdc12p are differences in interactions with the proline-rich sequences in the FH1 domain of Cdc12p and wider "wings" that interact with actin.
