Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2000 Jan 24;148(2):363-73.
doi: 10.1083/jcb.148.2.363.

Direct involvement of yeast type I myosins in Cdc42-dependent actin polymerization

T Lechler  1 , A Shevchenko, R Li
Affiliations

Direct involvement of yeast type I myosins in Cdc42-dependent actin polymerization

T Lechler et al. J Cell Biol. .

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.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Type I myosins associate with Bee1p. (A) High speed extracts were prepared from a wild-type strain and a strain carrying Bee1–protein A under the control of the BEE1 promoter. Ammonium sulfate was added to 30% saturation, and precipitated proteins were collected, resuspended, and bound to IgG–Sepharose. After extensive washing, proteins were eluted with 0.5 M acetic acid, pH 3.6. Shown is part of a Coomassie blue–stained gel that contains the indicated proteins: M, protein molecular weight markers; C, control (untagged wild-type extract); B, Bee1–protein A extract. The identity of proteins determined by mass spectroscopy analysis is indicated. (B) Extracts were prepared from strains carrying Myo3-HA under the control of the MYO3 promoter in either wild-type or Δvrp1 backgrounds. Immunoprecipitation was performed with anti-HA or anti-myc (control immunoprecipitate) coupled protein A beads. After washing and elution, proteins were subjected to SDS-PAGE, transferred to nitrocellulose, and blotted with the anti-Bee1p antibody ( Winter et al. 1999a). The extract lane is loaded with 5% of the input extract.
Figure 2
Figure 2
Type I myosins are necessary for cortical actin polymerization. (A) Examples of positive (left panel, wild-type extract), and negative (right panel, myo3G132R mutant extract) results for the reconstitution of actin assembly sites in permeabilized yeast cells. Assay conditions are described in Materials and Methods. (B) Concentrated extracts were prepared from various strains as indicated below the histograms and assayed for their ability to restore actin assembly activity to urea-treated permeabilized yeast cells. Quantitation of cells that assembled rhodamine actin into the bud was as described ( Lechler and Li 1997). For immunodepletion, anti-myc (mock depletion) or anti-HA coupled protein A beads were added to concentrated extracts prepared from a strain that expresses Myo3-HA in the Δmyo3 Δmyo5 background. After incubation, beads were pelleted and supernatants were removed for analysis. Gel inset above corresponding lanes of the histogram, shows depletion of Myo3-HA, but not Bee1p by the anti-HA beads. (C) Extracts from wild-type and Δmyo3 Δmyo5 cells were mixed at the indicated ratios and then assayed for their ability to restore actin assembly in urea-treated permeabilized cells. The complementation efficiency was quantified and plotted against the extract ratios. Each data point is an average of duplicate reactions.
Figure 3
Figure 3
Myosin motor activity is required for actin assembly. Reconstitution was performed with buffer or extracts as listed in the first line under the histogram. Polymerization was performed in the presence of 1 mM ATP (−), 1 mM ATP plus 20 mM BDM, or with 1 mM AMPPNP (with no ATP), as indicated below the histogram. Reconstitution procedure and quantitation of the results were performed as described ( Lechler and Li 1997).
Figure 4
Figure 4
Phosphorylation of type I myosins is an important step in the stimulation of actin polymerization by Cdc42p. (A) Reconstitution of actin assembly sites was carried out with concentrated extracts prepared from strains carrying the myo3S357A and MYO3S357D mutants as their only source of type I myosins. Reconstitution procedure and quantitation of the results were performed as described ( Lechler and Li 1997). AP: alkaline phosphatase used for treating wild-type or MYO3S357D mutant extracts (see text). (B) A mutant Myo3p mimicking constitutive phosphorylation rescues the actin assembly defect of a temperature-sensitive CDC42 allele. Wild-type, cdc42-1, and cdc42-1 cells expressing MYO3S357D or myo3S357A under the endogenous MYO3 promoter were grown at room temperature, shifted to 34°C for 2 h, permeabilized, and subjected to rhodamine actin assembly assays as described ( Li et al. 1995).
Figure 5
Figure 5
Type I myosins interact with the Arp2/3 complex. (A) Alignment of the COOH-terminal acidic domains of Myo3p, Myo5p, and Bee1p. Residues conserved in all three are in bold. (B) GST fusions to the COOH-terminal 38 aa of Myo3p and the COOH-terminal 54 aa of Myo5p and Bee1p were purified from bacteria. Fusion proteins bound to glutathione-agarose beads were incubated with 50 nM Arp2/3 complex purified as described ( Winter et al. 1999b). After washing, bound proteins were eluted into SDS-PAGE sample buffer. Upper panel, anti-Arp2p immunoblot; lower panel, Coomassie-stained gel showing that equal amounts of GST fusion proteins were added. (C) Extracts at 15 mg/ml were prepared from strains carrying Myo3-HA or Myo3ΔA-HA (Myo3p lacking the COOH-terminal acidic domain) in the Δmyo3 Δmyo5 background. Anti-myc (control immunoprecipitate) and anti-HA immunoprecipitations were performed, and bound proteins were analyzed by immunoblotting with anti-Arp2p antibodies. Extracts used are listed below the gel. The extract lane shows the presence of Arp2p in preimmunoprecipitate extracts.
Figure 6
Figure 6
The type I myosin and Bee1p acidic tails have redundant functions. (A) Equal concentration of cells from strains as indicated were spotted onto yeast extract, peptone, dextrose (YPD) plates, grown for 36 h, and then photographed. myo5ΔA Δmyo3: a strain expressing myo5ΔA in the Δmyo3 Δmyo5 double mutant background; bee1ΔA myo5ΔA Δmyo3: a strain expressing bee1ΔA and myo5ΔA in the Δmyo3 Δmyo5 double mutant background. (B) Rhodamine-phalloidin staining (upper panels) and differential interference contrast (DIC) images (lower panels) of wild-type and bee1ΔA myo5ΔA Δmyo3 cells grown at room temperature.
Figure 7
Figure 7
Bee1p and type I myosin acidic tails function redundantly in actin assembly. (A) Concentrated extracts were prepared from the strains as indicated and tested for their ability to restore the actin assembly activity of urea-treated permeabilized yeast cells. Reconstitution procedure and quantitation of the results were performed as described ( Lechler and Li 1997). (B) Extracts from wild-type and bee1ΔA myo5ΔA Δmyo3 cells were mixed at the indicated ratios on the horizontal axis and then assayed for their ability to restore actin assembly in urea-treated permeabilized cells. The complementation efficiency was quantified and plotted against the extract ratios. Each data point is an average of duplicate reactions.
See this image and copyright information in PMC

Comment in

  • The tails of two myosins.
    Machesky LM. Machesky LM. J Cell Biol. 2000 Jan 24;148(2):219-21. doi: 10.1083/jcb.148.2.219. J Cell Biol. 2000. PMID: 10648552 Free PMC article. No abstract available.

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Adams A.E., Johnson D.I., Longnecker R.M., Sloat B.F., Pringle J.R. CDC42 and CDC43, two additional genes involved in budding and the establishment of cell polarity in the yeast Saccharomyces cerevisiae . J. Cell Biol. 1990;111:131–142 . - PMC - PubMed
    1. Adams R.J., Pollard T.D. Propulsion of organelles isolated from Acanthamoeba along actin filaments by myosin-I. Nature. 1986;322:754–756 . - PubMed
    1. Adams R.J., Pollard T.D. Binding of myosin I to membrane lipids. Nature. 1989;340:565–568 . - PubMed
    1. Anderson B.L., Boldogh I., Evangelista M., Boone C., Greene L.A., Pon L.A. The Src homology domain 3 (SH3) of a yeast type I myosin, Myo5p, binds to verprolin and is required for targeting to sites of actin polymerization. J. Cell Biol. 1998;141:1357–1370 . - PMC - PubMed
    1. Aspenstrom P., Lindberg U., Hall A. Two GTPases, Cdc42 and Rac, bind directly to a protein implicated in the immunodeficiency disorder Wiskott-Aldrich syndrome. Curr. Biol. 1996;6:70–75 . - PubMed

Publication types

MeSH terms

Substances