Reconstitution of Human Arp2/3 Complex Reveals Critical Roles of Individual Subunits in Complex Structure and Activity

Gournier, Helene; Goley, Erin D.; Niederstrasser, Hanspeter; Trinh, T. Minh-ha; Welch, Matthew D.

doi:10.1016/s1097-2765(01)00393-8

Cited by 156 publications

(188 citation statements)

References 41 publications

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“…A function in VCA binding and in tethering Arp2 to the complex could both explain a loss of actin nucleation in ⌬arc40 extract. Our result is also largely consistent with the results from reconstitution experiments using human Arp2/3 subunit expressed in insect cells (33). Partial complex was purified from a reconstitution mixture that lacked both ARPC1 and ARPC5.…”

Section: Arpc1/arc40 Functions In Budding Yeastsupporting

confidence: 89%

ARPC1/Arc40 Mediates the Interaction of the Actin-related Protein 2 and 3 Complex with Wiskott-Aldrich Syndrome Protein Family Activators

Pan

Égile

Lipkin

et al. 2004

Journal of Biological Chemistry

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The actin-related protein 2 and 3 (Arp2/3) complex is a seven-subunit protein complex that nucleates actin filaments at the cell cortex. Despite extensive cross-linking, crystallography, genetic and biochemical studies, the contribution of each subunit to the activity of the complex remains largely unclear. In this study we characterized the function of the 40-kDa subunit, ARPC1/ Arc40, of the yeast Arp2/3 complex. We showed that this subunit is indeed a stable component of the Arp2/3 complex, but its highly unusual electrophoretic mobility eluded detection in previous studies. Recombinant Arc40 bound the VCA domain of Wiskott-Aldrich syndrome protein family activators at a K d of 0.45 M, close to that of the full complex with VCA (0.30 M), and this interaction was dependent on the conserved tryptophan at the COOH terminus of VCA. Using a newly constructed ⌬arc40 yeast strain, we showed that loss of Arc40 severely reduced the binding affinity of the Arp2/3 complex with VCA as well as the nucleation activity of the complex, suggesting that Arc40 contains an important contact site of the Arp2/3 complex with VCA. The ⌬arc40 cells exhibited reduced growth rate, loss of actin patches, and accumulation of cables like actin aggregates, phenotypes typical of other subunit nulls, suggesting that Arc40 functions exclusively within the Arp2/3 complex.

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Section: Arpc1/arc40 Functions In Budding Yeastsupporting

confidence: 89%

ARPC1/Arc40 Mediates the Interaction of the Actin-related Protein 2 and 3 Complex with Wiskott-Aldrich Syndrome Protein Family Activators

Pan

Égile

Lipkin

et al. 2004

Journal of Biological Chemistry

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“…Intensive studies had revealed that VCA's ability to bind monomer actin through its V subdomain is critical for actin nucleation (Miki and Takenawa, 1998). The CA subdomain confers to N-WASP its binding ability to the Arp2/3 complex as evidenced by physicochemical assays (Machesky and Insall, 1998;Marchand et al, 2001) and x-ray crystallography and cross-linking experiments (Gournier et al, 2001;Robinson et al, 2001;Zalevsky et al, 2001). Actin polymerization, nucleation, and branching are enhanced in the presence of VCA protein in vitro Machesky et al, 1999;Rohatgi et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

Inhibiting the Arp2/3 Complex Limits Infection of Both Intracellular Mature Vaccinia Virus and Primate Lentiviruses

Komano

Miyauchi

Matsuda

et al. 2004

MBoC

100

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Characterizing cellular factors involved in the life cycle of human immunodeficiency virus type 1 (HIV-1) is an initial step toward controlling replication of HIV-1. Actin polymerization mediated by the Arp2/3 complex has been found to play a critical role in some pathogens' intracellular motility. We have asked whether this complex also contributes to the viral life cycles including that of HIV-1. We have used both the acidic domains from actin-related protein (Arp) 2/3 complex-binding proteins such as the Wiscott-Aldrich syndrome protein (N-WASP) or cortactin, and siRNA directing toward Arp2 to inhibit viral infection. HIV-1, simian immunodeficiency virus (SIV), and intracellular mature vaccinia virus (IMV) were sensitive to inhibition of the Arp2/3 complex, whereas MLV, HSV-1, and adenovirus were not. Interestingly, pseudotyping HIV-1 with vesicular stomatitis virus G protein (VSV-G) overcame this inhibition. Constitutive inhibition of the Arp2/3 complex in the T-cell line H9 also blocked replication of HIV-1. These data suggested the existence of an Arp2/3 complex-dependent event during the early phase of the life cycles of both primate lentiviruses and IMV. Inhibiting the HIV-1's ability to activate Arp2/3 complex could be a potential chemotherapeutic intervention for acquired immunodeficiency syndrome (AIDS)

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“…More recent evidence suggests further that WASp binding may induce conformational rearrangements that propagate through all seven subunits of the Arp2/3 complex (5,6). The p35/ARPC2 and p19/ARPC4 subunits form the structural core of the complex and mediate binding to the side of the mother filament (3,(7)(8)(9)(10). Functions of the remaining three subunits have remained elusive.…”

mentioning

confidence: 99%

“…Two biochemical studies found that the Arp2/3 complex lacking p40/ARPC1 shows severely reduced actin nucleation activity in vitro (8,15). p40/ARPC1 also binds to the VCA (verprolin homology domain, connector, acidic) domain of S. cerevisiae WASp (Las17) (15,16) and directly contacts two other subunits in the complex, p19/ARPC4 and p15/ARPC5 (3).…”

mentioning

confidence: 99%

The p40/ARPC1 Subunit of Arp2/3 Complex Performs Multiple Essential Roles in WASp-regulated Actin Nucleation

Balcer

Daugherty-Clarke

Goode

2010

Journal of Biological Chemistry

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The Arp2/3 complex is a conserved seven-subunit actinnucleating 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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Reconstitution of Human Arp2/3 Complex Reveals Critical Roles of Individual Subunits in Complex Structure and Activity

Cited by 156 publications

References 41 publications

ARPC1/Arc40 Mediates the Interaction of the Actin-related Protein 2 and 3 Complex with Wiskott-Aldrich Syndrome Protein Family Activators

ARPC1/Arc40 Mediates the Interaction of the Actin-related Protein 2 and 3 Complex with Wiskott-Aldrich Syndrome Protein Family Activators

Inhibiting the Arp2/3 Complex Limits Infection of Both Intracellular Mature Vaccinia Virus and Primate Lentiviruses

The p40/ARPC1 Subunit of Arp2/3 Complex Performs Multiple Essential Roles in WASp-regulated Actin Nucleation

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