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

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 Here, we dissected the structure and function of Saccharomyces cerevisiae which is encoded by the essential ARC40 gene, by analyzing 39 integrated alleles that target its conserved surfaces. We identified three distinct sites on required for function in vivo: one site contacts one contacts and one site resides in an extended structural "arm" of p40/duanyu37C1. 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 specifically impaired WASp-induced nucleation. In contrast, lethal mutations at the contact with 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/duanyu37C1 arm domain to bind the VCA domain of WASp. Together, these data indicate that p40/duanyu37C1 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 2) propagation of WASp activation signals via contacts with and 3) direct facilitation of actin nucleation through interactions of the extended arm with the VCA domain of WASp.

KEYWORDS: {{ getKeywords(articleDetailText.words) }}