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Reference: Balcer HI, et al. (2010) The p40/ARPC1 Subunit of Arp2/3 Complex Performs Multiple Essential Roles in WASp-regulated Actin Nucleation. J Biol Chem 285(11):8481-91

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Abstract

The Arp2/3 complex is a conserved seven-subunit actin-nucleating machine activated by Wiskott Aldrich Syndrome protein (WASp). Despite its central importance in a broad range of cellular processes, many critical aspects of the mechanism of 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 S. 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 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 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.

Reference Type
Journal Article
Authors
Balcer HI, Daugherty-Clarke K, Goode BL
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