Abstract
Arp2/3 (actin-related protein 2/3) complex is a seven-subunit complex that nucleates branched actin filaments in response to cellular signals. Nucleation-promoting factors such as WASp/Scar family proteins activate the complex by facilitating the activating conformational change and recruiting the first actin monomer for the daughter branch. Here we address the role of the Arp2 subunit in the function of Arp2/3 complex by isolating a version of the complex lacking Arp2 (Arp2Delta Arp2/3 complex) from fission yeast. An x-ray crystal structure of the DeltaArp2 Arp2/3 complex showed that the rest of the complex is unperturbed by the loss of Arp2. However, the Arp2Delta Arp2/3 complex was inactive in actin nucleation assays, indicating that Arp2 is essential to form a branch. A fluorescence anisotropy assay showed that Arp2 does not contribute to the affinity of the complex for Wsp1-VCA, a Schizosaccharomyces pombe nucleation-promoting factor protein. Fluorescence resonance energy transfer experiments showed that the loss of Arp2 does not prevent VCA from recruiting an actin monomer to the complex. Truncation of the N terminus of ARPC5, the smallest subunit in the complex, increased the yield of Arp2Delta Arp2/3 complex during purification but did not compromise nucleation activity of the full Arp2/3 complex.
Highlights
26490 JOURNAL OF BIOLOGICAL CHEMISTRY called actin patches, which are sites of endocytosis located at cell poles during interphase and the cleavage furrow during cytokinesis [5,6,7]
A model built by fitting crystal structures into reconstructions of electron tomograms of branch junctions shows that all seven subunits of Arp2/3 complex contact the mother filament and that the barbed ends of Arp2 and Arp3 interact with the pointed end of the daughter filament [9]
The V region of nucleation promoting factors (NPFs) binds an actin monomer [13, 14], recruiting it to the branch point, whereas the C and A regions bind to Arp2/3 complex and are thought to facilitate conformational changes required for nucleation [14, 15]
Summary
Purification of S. pombe Arp2/3 Complex—We purified native Arp2/3 complex from Schizosaccharomyces pombe strain TM011 typically starting with 500 g of wet cells. Cells from an 8-liter culture were harvested and lysed in 300 ml of 20 mM Tris, pH 8.0, 25 mM NaCl, 2 mM DTT, and 1 mM phenylmethylsulfonyl fluoride containing four protease inhibitor tablets (Roche Applied Science) per 50 ml. Polymerization reactions of 100 l were assembled as follows: 2 l of 10 mM EGTA and 1 mM MgCl2 were added to 20 l of 20 M 15% pyrene actin monomers in G-buffer (2 mM Tris-HCl, pH 8.0, 0.2 mM ATP, 0.1 mM CaCl2, 0.5 mM DTT, and 0.01% NaN3) followed immediately by adding 78 l of a solution containing Arp2/3 complex, SpWsp1-VCA with buffers, and salts to bring the final reaction conditions to 10 mM imidazole, pH 7.0, 50 mM KCl, 1 mM EGTA, 1 mM MgCl2. Z-series were collected in 0.6-m steps with 200-ms sequential excitation of YFP and CFP
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