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

Abstract

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.

Role of ARPC2 in Human Gastric Cancer

“…As reported previously, Arp2/3 complex is essential for cell motility [11, 20]. As one of the subunits of Arp2/3, ARPC2 also promoted tumor development and progression.…”

Structural basis for regulation of Arp2/3 complex by GMF

“…Within one swath is a group of conserved basic residues that may interact with acidic residues in A. Some of these basic residues, either alone or in combination, have been shown to be important for viability of budding yeast, corroborating their functional relevance 36 . Also in the conserved swaths are candidate hydrophobic residues that could potentially interact with the conserved tryptophan in A, which is important for interaction of A with ARPC1 35 .…”

Arp2/3 complex is bound and activated by two WASP proteins

“…The proximity of this basic patch to ArpC5 could also explain the reported cross-linking of N-WASP VCA to this subunit of acanthamoeba Arp2/3 complex (25). Further, disruption of the acidic region binding surface may also explain the lethal phenotype of the Arc40-143 allele in budding yeast when this surface is mutated (35). As at the Arp3 site, we place the C region on Arp2 as a helix mimicking the interaction of the V region with actin (9,10,15,36).…”