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Reference: Daugherty KM and Goode BL (2008) Functional Surfaces on the p35/ARPC2 Subunit of Arp2/3 Complex Required for Cell Growth, Actin Nucleation, and Endocytosis. J Biol Chem 283(24):16950-9

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Abstract


The Arp2/3 complex is comprised of seven evolutionarily conserved subunits, and upon activation by WASp or another nucleation promoting factor (NPF) nucleates the formation of actin filaments. These events are critical for driving a wide range of cellular processes, including motility, endocytosis, and intracellular trafficking. However, an in depth understanding of the Arp2/3 complex activation and nucleation mechanism is still lacking. Here, we used a mutagenesis approach in S. cerevisiae to dissect the structural and functional roles of the p35/ARPC2 subunit. Using integrated alleles that target conserved and solvent-exposed residues, we identified surfaces on p35/ARPC2 required for cell growth, actin organization, and endocytosis. In parallel, we purified the mutant Arp2/3 complexes and compared their actin assembly activities both in the presence and absence of WASp. The majority of alleles with defects mapped to one face of p35/ARPC2, where there was a close correlation between loss of actin nucleation and endocytosis. A second site required for nucleation and endocytosis was identified near the contact surface between p35/ARPC2 and p19/ARPC4. A third site was identified at a more distal conserved surface, which was critical for endocytosis but not nucleation. These findings pinpoint the key surfaces on p35/ARPC2 required for Arp2/3 complex-mediated actin assembly and cellular function, and provide a higher resolution view of Arp2/3 structure and mechanism.

Reference Type
Journal Article
Authors
Daugherty KM, Goode BL
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