Dissection of Arp2/3 Complex Actin Nucleation Mechanism and Distinct Roles for Its Nucleation-Promoting Factors in Saccharomyces cerevisiae

Abstract

Actin nucleation by the Arp2/3 complex is under tight control, remaining inactive until stimulation by nucleation-promoting factors (NPFs). Although multiple NPFs are expressed in most cell types, little is known about how they are coordinated and whether they perform similar or distinct functions. We examined genetic relationships among the four S. cerevisiae NPFs. Combining las17D with pan1-101 or myo3Dmyo5D was lethal at all temperatures, whereas combining pan1-101 with myo3Dmyo5D showed no genetic interaction and abp1D partially suppressed las17D. These data suggest that NPFs have distinct and overlapping functions in vivo. We also tested genetic interactions between each NPF mutant and seven different temperature-sensitive arp2 alleles and purified mutant Arp2/3 complexes to compare their activities. Two arp2 alleles with mutations at the barbed end were severely impaired in nucleation, providing the first experimental evidence that Arp2 nucleates actin at its barbed end in vitro and in vivo.

The p40/ARPC1 Subunit of Arp2/3 Complex Performs Multiple Essential Roles in WASp-regulated Actin Nucleation

“…Accomplishing this required developing a new method for isolating mutant Arp2/3 complexes. Although small quantities of yeast Arp2/3 complex can be isolated from nonlethal yeast strains using a cleavable affinity tag integrated at the C terminus of any one of several different subunits (31), this established strategy precludes purification of Arp2/3 complex from lethal mutants. To overcome this obstacle, we developed a new purification strategy, which has four steps: 1) co-expression in a haploid strain of untagged mutant arc40 and HA-tagged wild type Arc40; this strain also has a TEV-3xHA tag integrated at the C terminus of Arp2; 2) co-isolation of mutant and wild type Arc40-containing Arp2/3 complexes on HA antibody-coated beads; 3) release of mutant Arp2/3 complex by TEV protease digestion; and 4) a final incubation with HA antibody-coated beads to clear any residual wild type Arp2/3 complex.…”

“…p40/ARPC1 Functional Roles in Actin Nucleation-To date, only a limited number of studies have used mutational analyses to dissect Arp2/3 complex structure and function (6,10,31,33). The wild type Arp2/3 complex exists in equilibrium among multiple conformation states, including inactive (open), intermediate, and "primed" (closed) states, with the primed state most closely resembling the conformation of the WASp-bound Arp2/3 complex (6).…”

“…A third study revealed that mutations at the barbed end of Arp2 (e.g. arp2-1 and arp2-2) strongly impair actin nucleation in both the presence and the absence of WASp (31). A fourth study dissected the structure and function of the p35/ARPC2 subunit and identified surfaces critical for actin nucleation and endocytosis (10).…”

“…4, C and D). These effects are reminiscent of arc35-5 and arp2-7 (6,31), suggesting that the interaction between p40/ARPC1 and p15/ARPC15 may help hold the Arp2/3 complex in an inactive state when NPFs are not present, suppressing nucleation. Because p15/ARPC5 physically interacts with p40/ARPC1 and Arp2, our data suggest that p15/ARPC5 may function as a molecular "latch," holding p40/ARPC1 and perhaps Arp2 in a less active position until WASp triggers conformational rearrangements in the complex that are propagated in part via p19/ ARPC4 interactions with p40/ARPC1.…”

Interaction of the Endocytic Scaffold Protein Pan1 with the Type I Myosins Contributes to the Late Stages of Endocytosis

“…It has been shown previously that the pan1-4 allele exhibits synthetic lethality when combined with sla1⌬ (Tang et al, 2000); however, this allele corresponds to an extensive C-terminal truncation lacking hundreds of amino acids (Zeng et al, 2001), whereas our truncation is missing just the final 170 amino acids and preserves the F-actin and acidic Arp2/3 binding sites. Perhaps the combination of losing regulation of both Las17 and the type I myosins is particularly deleterious; for example, the Arp2/3 stimulatory activity of Las17 is required in cells lacking the Arp2/3 activity of the type I myosins (Evangelista et al, 2000;D'Agostino and Goode, 2005). Likewise, loss of Las17 regulation by deleting SLA1, along with altered Myo3 or Myo5 regulation by deleting the PRD of Pan1 could lead to irrevocably perturbed actin structures.…”