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. 2010 Mar 12;285(11):8481-91.
doi: 10.1074/jbc.M109.054957. Epub 2010 Jan 12.

The p40/ARPC1 subunit of Arp2/3 complex performs multiple essential roles in WASp-regulated actin nucleation

Heath I Balcer  1 , Karen Daugherty-Clarke, Bruce L Goode
Affiliations

The p40/ARPC1 subunit of Arp2/3 complex performs multiple essential roles in WASp-regulated actin nucleation

Heath I Balcer et al. J Biol Chem. .

Abstract

The Arp2/3 complex is a conserved seven-subunit actin-nucleating machine activated by WASp (Wiskott Aldrich syndrome protein). Despite its central importance in a broad range of cellular processes, many critical aspects of the mechanism of the Arp2/3 complex have yet to be resolved. In particular, some of the individual subunits in the complex have not been assigned clear functional roles, including p40/ARPC1. Here, we dissected the structure and function of Saccharomyces cerevisiae p40/ARPC1, which is encoded by the essential ARC40 gene, by analyzing 39 integrated alleles that target its conserved surfaces. We identified three distinct sites on p40/ARPC1 required for function in vivo: one site contacts p19/ARPC4, one contacts p15/ARPC5, and one site resides in an extended structural "arm" of p40/ARPC1. Using a novel strategy, we purified the corresponding lethal mutant Arp2/3 complexes from yeast and compared their actin nucleation activities. Lethal mutations at the contact with p19/ARPC4 specifically impaired WASp-induced nucleation. In contrast, lethal mutations at the contact with p15/ARPC5 led to unregulated ("leaky") nucleation in the absence of WASp. Lethal mutations in the extended arm drastically reduced nucleation, and the same mutations disrupted the ability of the purified p40/ARPC1 arm domain to bind the VCA domain of WASp. Together, these data indicate that p40/ARPC1 performs at least three distinct, essential functions in regulating Arp2/3 complex-mediated actin assembly: 1) suppression of spontaneous nucleation by the Arp2/3 complex, which requires proper contacts with p15/ARPC5; 2) propagation of WASp activation signals via contacts with p19/ARPC2; and 3) direct facilitation of actin nucleation through interactions of the extended arm with the VCA domain of WASp.

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Figures

FIGURE 1.
FIGURE 1.
Alignment of p40/ARPC1 primary sequences from S. cerevisiae (S.c.), H. sapiens (H.s.), B. taurus (B.t.), and S. pombe (S.p.). Bovine structural elements are designated above the primary sequences. The arrows represent β-sheets, and boxes represent α-helices. The seven propeller blades of p40/ARPC1 are colored-coded: green (blade 1), blue (blade 2), pink (blade 3), cyan (blade 4), orange (blade 5), lavender (blade 6), and gray (blade 7), so that numbering is consistent with Fig. 2. Residues that are identical in all four species are shaded in dark gray; similar residues are shaded in light gray. Allele numbers and mutations are shown above the S. cerevisiae sequence. Sequences internally deleted in arc40Δ311–340 are marked by a red line. Residues included in the arm domain peptide (residues 306–355) are underlined in green.
FIGURE 2.
FIGURE 2.
Analysis of initial p40/ARPC1 alanine scan mutations. A, crystal structure of bovine Arp2/3 complex (adapted from Ref. 3), with the seven subunits differently colored: Arp2 (pink), Arp3 (orange), p40/ARPC1 (tan), p35/ARPC2 (cyan), p18/ARPC3 (purple), p19/ARPC4 (blue), and p15/ARPC5 (yellow). B, top view of p40/ARPC1 showing the positions of residues mutated in the initial alanine scan allele collection modeled on bovine ARPC1. The one temperature-sensitive allele (arc40-118; Fig. S1) is green; all other alleles were psuedo-wild type for cell growth and actin organization and are colored blue. The propeller blades are numbered 1–7 counterclockwise in the top view and color-coded to be consistent with Fig. 1. The side chains of residues mutated in arc40 alleles are shown. C, growth of integrated arc40 haploid strains at 25 °C. All of the strains were serially diluted and plated for growth on YPD medium at different temperatures (16, 25, 30, 34, and 37 °C); no defects in growth were observed at any temperature (not shown).
FIGURE 3.
FIGURE 3.
Analysis of lethal arc40 alleles. S. cerevisiae numbering of residues is used in C–F. All of the subunits in the Arp2/3 complex are colored as in Fig. 2A. A, tetrad analysis of arc40 alleles integrated at the LEU2 locus in the heterozygous diploid strain arc40Δ::HIS3/ARC40. The diploid strains were sporulated, and tetrads were dissected. The specific patterns of growth for all alleles except ARC40 indicate lethality (see “Results”). B, cell growth phenotypes of an ARC40 strain and arc40Δ::HIS3 strains with integrated LEU2-marked copies of arc40Δ or specific arc40 alleles carrying a URA3 marked ARC40 plasmid. The cells were grown in YPD medium overnight and then serially diluted, plated on YPD, −uracil, −leucine, or 5-fluoroorotic acid-containing medium, and grown for 3 days at 25 °C. C, surface rendered structure of p40/ARPC1 (tan) showing its contacts with p19/ARPC4 (stick model with green mesh). Residues mutated in lethal arc40-139 are highlighted in red and numbered. D, surface rendered structure of p40/ARPC1 showing contacts with p15/ARPC5, with residues in lethal arc40-141 highlighted in red. Residues in nonlethal arc40-142 are highlighted in purple. E, surface rendered structure of p40/ARPC1 (tan) with residues in lethal arc40-140 highlighted in red. F, surface rendered structure of p40/ARPC1 (tan) displaying residues that have been predicted to mediate mother filament side binding (17). Mutated residues that when combined yielded a lethal allele (arc40-143) are highlighted in red. Residues that when combined were pseudo-wild type are highlighted in purple.
FIGURE 4.
FIGURE 4.
Actin nucleation activities of wild type and arc40-139, arc40-141, and arc40-140 Arp2/3 complexes. The rates of assembly were determined from the slopes of the curves at their steepest points, which was between 25 and 50% assembly. A, C, and E, actin assembly reactions containing 3 μm monomeric rabbit muscle actin (5% pyrene-labeled) with variable concentrations of wild type (blue) or mutant (red) Arp2/3 complex. B, D, and F, actin assembly reactions containing 2 μm monomeric rabbit muscle actin (5% pyrene-labeled), Las17/WASp (5 nm for arc40-139, arc40-141 and 10 nm for arc40-140), and variable concentrations of wild type (blue) or mutant (red) Arp2/3 complex. A higher concentration of monomeric actin was used for the reactions in A, C, and E compared with B, D, and F to more readily detect base-line Arp2/3 complex nucleation activity in the absence of Las17/WASp.
FIGURE 5.
FIGURE 5.
The p40/ARPC1 “arm” domain binds to the VCA domain of Las17/WASp. A, 2 μm soluble Arc40 arm was incubated for 10 min with control beads or beads coated with full-length Las17/WASp or its VCA domain. The beads were pelleted, and the percentage of Arc40 arm bound was determined by analyzing pellets and supernatants on Coomassie-stained gels. B, wild type and mutant (arc40-140) GST-Arc40 arm peptides were immobilized on glutathione-agarose beads and incubated with 100 nm soluble, full-length Las17/WASp. The beads were pelleted, and the percentage of Las17/WASp bound was determined by analysis of the supernatant fractions by immunoblotting with anti-VCA antibodies. C, 75 nm wild type ARC40, arc40Δ311–340, or arc40-140 Arp2/3 complex was incubated with GST-VCA coated beads. The beads were pelleted, and the relative amounts of Arp2/3 complex bound were determined by analysis of the pellet fractions by immunoblotting with anti-Arp2 antibodies.
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