Surprisingly, we find that only ARC40 is fully essential for cell viability

Surprisingly, we find that only ARC40 is fully essential for cell viability. 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. A complex containing two actin-related proteins, Arp2p and Arp3p, has recently emerged as a strong candidate for nucleating actin assembly that drives the motility of the pathogenic bacterium (1, 2). This complex, termed the Arp2/3 complex, contains seven subunits conserved among eukaryotes and localizes to regions of actin-based motility, such as the actin comet tails of (1), and the leading edges of and fibroblasts (3C5). Biochemical studies have shown it to bind both pointed ends and sides of actin filaments to create T structures resembling the brush-like actin structures seen at the leading Ctsk edges of fish keratocytes (6, 7). Furthermore, the complex has a weak intrinsic actin nucleation activity that is significantly stimulated by the ActA protein of function of Arp2/3 complex. In fission yeast, Arp3p is an essential actin-patch component that functions to promote cell cycle-specific actin rearrangements (14). Sop2p, the fission yeast homolog of the 40-kDa subunit (p40) of the Arp2/3 complex is PI-103 Hydrochloride an essential protein that interacts with Arp3p but localizes to filamentous structures distinct from actin patches (15). In budding yeast, Arp2p and Arp3p have been shown to be components of actin patches (16, 17), the highly motile actin-rich structures that accumulate at sites of polarized growth during the yeast cell cycle. Both Arp2p and Arp3p function to maintain the proper organization of actin patches, and Arp3p is required for the motility of actin patches (16, 17). An Arp2p- and Arp3p-containing complex purified from budding yeast contained PI-103 Hydrochloride six equal stoichiometric subunits (17). Sequence identification of PI-103 Hydrochloride these subunits showed that they are highly conserved with the subunits of the human Arp2/3 complex (5). The only subunit missing in the purified budding yeast complex was p40. A homolog of p40, termed to be fully essential in our strain background. Deletion of genes encoding the other subunits PI-103 Hydrochloride gave rise to viable strains with varying degrees of growth defects, permitting us to analyze their relative roles in maintaining actin organization and the integrity of the Arp2/3 complex. MATERIALS AND METHODS Gene Disruption of Arp2/3 Complex Subunits. A heterozygous gene disruption strain (RLY180) was generated as described (17). The gene was PCR-amplified from genomic DNA by using primers SRp1 (5-GCG CGC CTG TGA TAT GTA TAT TTG TT-3) and SRp2 (5-GCG CGC CTA TCC TCT AAC GGC GCT CA-3) and cloned into pBluescript II SK(+) (Stratagene) by using ORF. These sites were blunted, and the gene from YDp-W (18), was inserted to generate the gene disruption plasmid pDW3. pDW3 was cut with gene disruption was confirmed by using PCR and restriction digest analysis (data not shown). The gene was amplified from genomic DNA by using primers DWp1 (5-GCG CGCTGCTA GTC AAT AAA AAC AC-3) and cloned into pSK+ by using ORF, which was replaced by the gene from YDp-W as above to generate pDW22. To generate the strain, pDW22 was cut with gene disruption was confirmed by using PCR (data not shown). genes were disrupted by using the one-step PCR-based method described (16). For deletion, a PCR fragment carrying the marker was amplified from pRS304 (19) with primers DWp19 (5-CAG AGA AGA CTC AAC ACA ACA CAC GCG AAC GAT CAA.