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.
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24 January 2000
Article|
January 24 2000
Direct Involvement of Yeast Type I Myosins in Cdc42-Dependent Actin Polymerization
Terry Lechler,
Terry Lechler
aDepartment of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115
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Anna Shevchenko,
Anna Shevchenko
bPeptide and Protein Group, European Molecular Biology Laboratory (EMBL), 69012 Heidelberg, Germany
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Andrej Shevchenko,
Andrej Shevchenko
bPeptide and Protein Group, European Molecular Biology Laboratory (EMBL), 69012 Heidelberg, Germany
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Rong Li
Rong Li
aDepartment of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115
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Terry Lechler
aDepartment of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115
Anna Shevchenko
bPeptide and Protein Group, European Molecular Biology Laboratory (EMBL), 69012 Heidelberg, Germany
Andrej Shevchenko
bPeptide and Protein Group, European Molecular Biology Laboratory (EMBL), 69012 Heidelberg, Germany
Rong Li
aDepartment of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115
Abbreviations used in this paper: aa, amino acid(s); AMPPNP, adenosine 5′-[beta, gamma-imido]triphosphate; Arp, actin-related protein; BDM, 2,3-butanedione 2-monoxime; GST, glutathione S-transferase; HA, hemagglutinin; N-WASP, neuronal WASP; PAK, p21-activated kinase; SH3, Src homology domain 3; WASP, Wiskott-Aldrich syndrome protein.
Received:
October 06 1999
Revision Requested:
December 02 1999
Accepted:
December 06 1999
Online ISSN: 1540-8140
Print ISSN: 0021-9525
© 2000 The Rockefeller University Press
2000
The Rockefeller University Press
J Cell Biol (2000) 148 (2): 363–374.
Article history
Received:
October 06 1999
Revision Requested:
December 02 1999
Accepted:
December 06 1999
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Citation
Terry Lechler, Anna Shevchenko, Andrej Shevchenko, Rong Li; Direct Involvement of Yeast Type I Myosins in Cdc42-Dependent Actin Polymerization. J Cell Biol 24 January 2000; 148 (2): 363–374. doi: https://doi.org/10.1083/jcb.148.2.363
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