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A two-tiered mechanism by which Cdc42 controls the localization and activation of an Arp2/3-activating motor complex in yeast.

Lechler T, Jonsdottir GA, Klee SK, Pellman D, Li R - J. Cell Biol. (2001)

Bottom Line: One branch, which requires formin homologues, mediates the recruitment of the Bee1p complex to the cortical site where the activated Cdc42p resides.The other is mediated by the p21-activated kinases, which activate the motor activity of myosin-I through phosphorylation.Together, these findings provide insights into the essential processes leading to polarization of the actin cytoskeleton.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
The establishment of cell polarity in budding yeast involves assembly of actin filaments at specified cortical domains. Elucidation of the underlying mechanism requires an understanding of the machinery that controls actin polymerization and how this machinery is in turn controlled by signaling proteins that respond to polarity cues. We showed previously that the yeast orthologue of the Wiskott-Aldrich Syndrome protein, Bee1/Las17p, and the type I myosins are key regulators of cortical actin polymerization. Here, we demonstrate further that these proteins together with Vrp1p form a multivalent Arp2/3-activating complex. During cell polarization, a bifurcated signaling pathway downstream of the Rho-type GTPase Cdc42p recruits and activates this complex, leading to local assembly of actin filaments. One branch, which requires formin homologues, mediates the recruitment of the Bee1p complex to the cortical site where the activated Cdc42p resides. The other is mediated by the p21-activated kinases, which activate the motor activity of myosin-I through phosphorylation. Together, these findings provide insights into the essential processes leading to polarization of the actin cytoskeleton.

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Bee1p, Vrp1p, and type I myosins form a complex that contains two activators of the Arp2/3 complex. (A) Bee1p and Vrp1p exist in a stable protein complex. Bee1p and Vrp1p in a native extract were analyzed on a Superose-6 gel filtration column. The positions of markers, blue dextran (2000 kD) and thyroglobulin (670 kD), are indicated. (B) Activation of the Arp2/3 complex by combined action of the A domain of Myo3p and the WH2 domain of Vrp1p. Pyrene actin assembly assays were performed at 1.75 μM actin, 0.25 μM Arp2/3, and 0.5 μM activator. (a) Activation of Arp2/3 by chimeric proteins containing the WH2 domain of Bee1p or Vrp1p fused to the A domain of Myo3p. The graphs are as follows: actin alone (no symbol); actin with Arp2/3 complex (○); actin with Arp2/3 complex and WH2(Bee1p)-A(Bee1p) (□); actin with Arp2/3 complex and WH2(Vrp1p)-A(Myo3p) (▪); and actin with Arp2/3 complex and WH2(Bee1p)-A(Myo3p) (•). (b) Activation of Arp2/3 complex by a dimer of WH2(Vrp1p) and A(Myo3p). The graph shows the following: actin alone (no symbol); actin with Arp2/3 complex (○); actin with Arp2/3 complex and WH2-A(Bee1p) (□); and actin with Arp2/3 complex, WH2(Vrp1p)-FRB, A(Myo3p)-FKBP12 with (▴) and without (♦) rapamycin.
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fig1: Bee1p, Vrp1p, and type I myosins form a complex that contains two activators of the Arp2/3 complex. (A) Bee1p and Vrp1p exist in a stable protein complex. Bee1p and Vrp1p in a native extract were analyzed on a Superose-6 gel filtration column. The positions of markers, blue dextran (2000 kD) and thyroglobulin (670 kD), are indicated. (B) Activation of the Arp2/3 complex by combined action of the A domain of Myo3p and the WH2 domain of Vrp1p. Pyrene actin assembly assays were performed at 1.75 μM actin, 0.25 μM Arp2/3, and 0.5 μM activator. (a) Activation of Arp2/3 by chimeric proteins containing the WH2 domain of Bee1p or Vrp1p fused to the A domain of Myo3p. The graphs are as follows: actin alone (no symbol); actin with Arp2/3 complex (○); actin with Arp2/3 complex and WH2(Bee1p)-A(Bee1p) (□); actin with Arp2/3 complex and WH2(Vrp1p)-A(Myo3p) (▪); and actin with Arp2/3 complex and WH2(Bee1p)-A(Myo3p) (•). (b) Activation of Arp2/3 complex by a dimer of WH2(Vrp1p) and A(Myo3p). The graph shows the following: actin alone (no symbol); actin with Arp2/3 complex (○); actin with Arp2/3 complex and WH2-A(Bee1p) (□); and actin with Arp2/3 complex, WH2(Vrp1p)-FRB, A(Myo3p)-FKBP12 with (▴) and without (♦) rapamycin.

Mentions: The approach that we have taken to understand the link between Cdc42 and actin polymerization is to first identify protein factors that are critical for actin polymerization and then determine how these proteins are controlled by Cdc42p during cell polarization. Previous studies identified two interacting proteins important for actin patch assembly and organization, Bee1p and Vrp1p, a homologue of mammalian WASP-interacting protein (WIP) (Lechler and Li, 1997; Ramesh et al., 1997; Vaduva et al., 1997; Naqvi et al., 1998). To determine whether Vrp1p and Bee1p actually form a stable biochemical association in vivo, we constructed a strain that expresses Vrp1p tagged at the COOH terminus with (myc)6 epitope under the native VRP1 promoter as the sole source of Vrp1p. Gel filtration analysis indicated that Bee1p and Vrp1p cofractionate completely in a protein complex that migrates at a rate corresponding to a 1,000 kD globular protein (Fig. 1 A). Coimmunoprecipitation experiments showed that the Bee1p–Vrp1p interaction is resistant to 1 M KCl (unpublished data). This result suggest that Bee1p and Vrp1p exist in vivo as a stable protein complex.


A two-tiered mechanism by which Cdc42 controls the localization and activation of an Arp2/3-activating motor complex in yeast.

Lechler T, Jonsdottir GA, Klee SK, Pellman D, Li R - J. Cell Biol. (2001)

Bee1p, Vrp1p, and type I myosins form a complex that contains two activators of the Arp2/3 complex. (A) Bee1p and Vrp1p exist in a stable protein complex. Bee1p and Vrp1p in a native extract were analyzed on a Superose-6 gel filtration column. The positions of markers, blue dextran (2000 kD) and thyroglobulin (670 kD), are indicated. (B) Activation of the Arp2/3 complex by combined action of the A domain of Myo3p and the WH2 domain of Vrp1p. Pyrene actin assembly assays were performed at 1.75 μM actin, 0.25 μM Arp2/3, and 0.5 μM activator. (a) Activation of Arp2/3 by chimeric proteins containing the WH2 domain of Bee1p or Vrp1p fused to the A domain of Myo3p. The graphs are as follows: actin alone (no symbol); actin with Arp2/3 complex (○); actin with Arp2/3 complex and WH2(Bee1p)-A(Bee1p) (□); actin with Arp2/3 complex and WH2(Vrp1p)-A(Myo3p) (▪); and actin with Arp2/3 complex and WH2(Bee1p)-A(Myo3p) (•). (b) Activation of Arp2/3 complex by a dimer of WH2(Vrp1p) and A(Myo3p). The graph shows the following: actin alone (no symbol); actin with Arp2/3 complex (○); actin with Arp2/3 complex and WH2-A(Bee1p) (□); and actin with Arp2/3 complex, WH2(Vrp1p)-FRB, A(Myo3p)-FKBP12 with (▴) and without (♦) rapamycin.
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Related In: Results  -  Collection

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fig1: Bee1p, Vrp1p, and type I myosins form a complex that contains two activators of the Arp2/3 complex. (A) Bee1p and Vrp1p exist in a stable protein complex. Bee1p and Vrp1p in a native extract were analyzed on a Superose-6 gel filtration column. The positions of markers, blue dextran (2000 kD) and thyroglobulin (670 kD), are indicated. (B) Activation of the Arp2/3 complex by combined action of the A domain of Myo3p and the WH2 domain of Vrp1p. Pyrene actin assembly assays were performed at 1.75 μM actin, 0.25 μM Arp2/3, and 0.5 μM activator. (a) Activation of Arp2/3 by chimeric proteins containing the WH2 domain of Bee1p or Vrp1p fused to the A domain of Myo3p. The graphs are as follows: actin alone (no symbol); actin with Arp2/3 complex (○); actin with Arp2/3 complex and WH2(Bee1p)-A(Bee1p) (□); actin with Arp2/3 complex and WH2(Vrp1p)-A(Myo3p) (▪); and actin with Arp2/3 complex and WH2(Bee1p)-A(Myo3p) (•). (b) Activation of Arp2/3 complex by a dimer of WH2(Vrp1p) and A(Myo3p). The graph shows the following: actin alone (no symbol); actin with Arp2/3 complex (○); actin with Arp2/3 complex and WH2-A(Bee1p) (□); and actin with Arp2/3 complex, WH2(Vrp1p)-FRB, A(Myo3p)-FKBP12 with (▴) and without (♦) rapamycin.
Mentions: The approach that we have taken to understand the link between Cdc42 and actin polymerization is to first identify protein factors that are critical for actin polymerization and then determine how these proteins are controlled by Cdc42p during cell polarization. Previous studies identified two interacting proteins important for actin patch assembly and organization, Bee1p and Vrp1p, a homologue of mammalian WASP-interacting protein (WIP) (Lechler and Li, 1997; Ramesh et al., 1997; Vaduva et al., 1997; Naqvi et al., 1998). To determine whether Vrp1p and Bee1p actually form a stable biochemical association in vivo, we constructed a strain that expresses Vrp1p tagged at the COOH terminus with (myc)6 epitope under the native VRP1 promoter as the sole source of Vrp1p. Gel filtration analysis indicated that Bee1p and Vrp1p cofractionate completely in a protein complex that migrates at a rate corresponding to a 1,000 kD globular protein (Fig. 1 A). Coimmunoprecipitation experiments showed that the Bee1p–Vrp1p interaction is resistant to 1 M KCl (unpublished data). This result suggest that Bee1p and Vrp1p exist in vivo as a stable protein complex.

Bottom Line: One branch, which requires formin homologues, mediates the recruitment of the Bee1p complex to the cortical site where the activated Cdc42p resides.The other is mediated by the p21-activated kinases, which activate the motor activity of myosin-I through phosphorylation.Together, these findings provide insights into the essential processes leading to polarization of the actin cytoskeleton.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
The establishment of cell polarity in budding yeast involves assembly of actin filaments at specified cortical domains. Elucidation of the underlying mechanism requires an understanding of the machinery that controls actin polymerization and how this machinery is in turn controlled by signaling proteins that respond to polarity cues. We showed previously that the yeast orthologue of the Wiskott-Aldrich Syndrome protein, Bee1/Las17p, and the type I myosins are key regulators of cortical actin polymerization. Here, we demonstrate further that these proteins together with Vrp1p form a multivalent Arp2/3-activating complex. During cell polarization, a bifurcated signaling pathway downstream of the Rho-type GTPase Cdc42p recruits and activates this complex, leading to local assembly of actin filaments. One branch, which requires formin homologues, mediates the recruitment of the Bee1p complex to the cortical site where the activated Cdc42p resides. The other is mediated by the p21-activated kinases, which activate the motor activity of myosin-I through phosphorylation. Together, these findings provide insights into the essential processes leading to polarization of the actin cytoskeleton.

Show MeSH
Related in: MedlinePlus