Genetic Dissection of the Budding Yeast Arp2/3 Complex: A Comparison of the in vivo and Structural Roles of Individual Subunits

• Published in: Proceedings of the National Academy of Sciences - PNAS 1999-06, Vol.96 (13), p.7288-7293
• Main Authors: Winter, Dirk C., Choe, Elizabeth Y., Li, Rong
• Format: Article
• Language: English
• Subjects: Acanthamoeba; Actin-Related Protein 2; Actin-Related Protein 3; Actins; Actins - chemistry; Actins - genetics; Actins - metabolism; Animals; Antibodies; Base Sequence; Biological Sciences; Cells; Cellular biology; Cytoskeletal Proteins; Fungal Proteins - chemistry; Fungal Proteins - genetics; Fungal Proteins - metabolism; Genes; Genes, Fungal; Genetic mutation; Genetics; Humans; Molecular Sequence Data; Monomers; Mother cells; Polymerase chain reaction; Protein Binding; Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomycetales; Sequence Analysis; Sequence Deletion; Structure-Activity Relationship; Viability; Yeast; Yeasts
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.96.13.7288

In previous work, we identified the yeast Arp2/3 complex, which localizes to cortical actin patches and is required for their motility and integrity in vivo. This complex contains proteins homologous to each subunit of the Acanthamoeba and human Arp2/3 complex except for a 40-kDa subunit (p40), which was missing from the purified yeast complex. Here, we demonstrate by using immunoprecipitation and gel-filtration analysis that Arc40p, the homolog of p40 identified from the yeast genome database, associates with the yeast Arp2/3 complex. We have carried out gene disruptions of each subunit of the yeast Arp2/3 complex to study each subunit's role in the function of the complex. Surprisingly, we find that only ARC40 is fully essential for cell viability. Strains lacking each of the other subunits exhibit varying degrees of defects in cell growth and viability and in assembly and polarization of cortical actin patches. We have also examined each subunit's role in maintaining the structural integrity of the Arp2/3 complex. Arp2p, Arp3p, and Arc40p fall into the monomer pool in Δ arc19 and Δ arc35 cells, suggesting that Arc19p and Arc35p are the central scaffolding components of the complex. Arp2p and Arp3p do not have major roles in maintaining complex integrity, and Arc15p is required for association of Arp2p and Arc40p, but not other subunits, with the complex. These results provide evidence that each subunit contributes differently to the assembly and function of the Arp2/3 complex.