Functional surfaces on the p35/ARPC2 subunit of Arp2/3 complex required for cell growth, actin nucleation, and endocytosis.
The Arp2/3 complex is comprised of seven evolutionarily conserved subunits and upon activation by WASp or another nucleation promoting factor 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 Saccharomyces 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 in the 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.
Daugherty
Karen M
KM
Department of Biochemistry, Brandeis University and the Rosenstiel Basic Medical Sciences Center, Waltham, Massachusetts 02454, USA.
Goode
Bruce L
BL
D051376:Actin-Related Protein 2-3 Complex / Q000737:chemistry* / Q000378:metabolism; D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000483:Alleles; D000595:Amino Acid Sequence; D001665:Binding Sites; D049109:Cell Proliferation; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis; D008954:Models, Biological; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
D051376:Actin-Related Protein 2-3 Complex; D000199:Actins