Direct Involvement of Yeast Type I Myosins in Cdc42-Dependent Actin Polymerization

Abstract

The generation of cortical actin filaments is necessary for processes such as cell motility and cell polarization. Several recent studies have demonstrated that Wiskott-Aldrich syndrome protein (WASP) family proteins and the actin-related protein (Arp) 2/3 complex are key factors in the nucleation of actin filaments in diverse eukaryotic organisms. To identify other factors involved in this process, we have isolated proteins that bind to Bee1p/Las17p, the yeast WASP-like protein, by affinity chromatography and mass spectroscopic analysis. The yeast type I myosins, Myo3p and Myo5p, have both been identified as Bee1p-interacting proteins. Like Bee1p, these myosins are essential for cortical actin assembly as assayed by in vitro reconstitution of actin nucleation sites in permeabilized yeast cells. Analysis using this assay further demonstrated that the motor activity of these myosins is required for the polymerization step, and that actin polymerization depends on phosphorylation of myosin motor domain by p21-activated kinases (PAKs), downstream effectors of the small guanosine triphosphatase, Cdc42p. The type I myosins also interact with the Arp2/3 complex through a sequence at the end of the tail domain homologous to the Arp2/3-activating region of WASP-like proteins. Combined deletions of the Arp2/3-interacting domains of Bee1p and the type I myosins abolish actin nucleation sites at the cortex, suggesting that these proteins function redundantly in the activation of the Arp2/3 complex.

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

“…MYO3 and MYO5 are the only genes encoding type I myosins in the Saccharomyces cerevisiae genome. Deletion of either gene does not affect growth; however, a double mutant is severely compromised (Goodson et al, 1996;Lechler et al, 2000). myo5⌬ cells, but not myo3⌬ cells, have a temperature-sensitive endocytosis defect, indicating that Myo5 may be more competent in endocytosis than Myo3 (Geli and Riezman, 1996).…”

“…Typically, myosins have N-terminal motor domains that bind actin in an ATP-dependent cycle and C-terminal tails that bind cargo through protein interaction domains. Type I myosins have an SH3 domain in their C-terminal tails, and fungal type I myosins also have an A domain at the extreme C-terminus that binds and activates the Arp2/3 complex (Geli and Riezman, 1996;Goodson et al, 1996;Evangelista et al, 2000;Lechler et al, 2000). MYO3 and MYO5 are the only genes encoding type I myosins in the Saccharomyces cerevisiae genome.…”

“…Both the motor and Arp2/3 activities of Myo5 have been implicated in completing the late stages of endocytosis (Sun et al, 2006). In support of their endocytic roles, the C-terminal tails of Myo3 and Myo5 interact with multiple endocytic proteins: Las17, a yeast homologue of Wiskcott Aldrich Syndrome protein (WASP) (Evangelista et al, 2000); Vrp1, a yeast homologue of human WASP-interacting protein (Anderson et al, 1998); and Arc19 and Arc40, components of the Arp2/3 actin nucleating complex (Evangelista et al, 2000;Lechler et al, 2000). Vrp1 is an activator of the Arp2/3 actin polymerization activity of type I myosins in both S. cerevisiae and Schizosaccharomyces pombe (Sirotkin et al, 2005;Sun et al, 2006).…”

Transcript Profiles of Candida albicans Cortical Actin Patch Mutants Reflect Their Cellular Defects: Contribution of the Hog1p and Mkc1p Signaling Pathways

“…Indeed, cortical actin patches are the actual sites of endocytosis (29). S. cerevisiae myosin I (Myo3/5p) and Sla2p have been shown to play important roles in organizing the actin cytoskeleton and mediating endocytosis by the cortical actin patches (18,28,33,34). In addition, C. albicans SLA2 and MYO5 are required for hyphal formation, MYO5 being the unique gene encoding myosin I, hereby designated Myo5p (5,41).…”

Verprolin: A cool set of actin‐binding sites and some very HOT prolines

“…For strong activity, Arp2/3 requires stimulation by nucleation promoting factors (NPFs) (15). Verprolin associates with several NPFs including Las17p (or Bee1p) (yeast WASP) (21) and type I myosins (7,15,(23)(24)(25)(26) (Fig. 1).…”