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Interactions with PIP2, ADP-actin monomers, and capping protein regulate the activity and localization of yeast twinfilin.

Palmgren S, Ojala PJ, Wear MA, Cooper JA, Lappalainen P - J. Cell Biol. (2001)

Bottom Line: A mutant twinfilin that does not interact with actin monomers in vitro no longer localizes to cortical actin patches when expressed in yeast, suggesting that the ability to interact with actin monomers may be essential for the localization of twinfilin.Twinfilin also interacts with phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2), and its actin monomer-sequestering activity is inhibited by PI(4,5)P2.Based on these results, we propose a model for the biological role of twinfilin as a protein that localizes actin monomers to the sites of rapid filament assembly in cells.

View Article: PubMed Central - PubMed

Affiliation: Program in Cellular Biotechnology, Institute of Biotechnology, FIN-00014 University of Helsinki, Helsinki, Finland.

ABSTRACT
Twinfilin is a ubiquitous actin monomer-binding protein that regulates actin filament turnover in yeast and mammalian cells. To elucidate the mechanism by which twinfilin contributes to actin filament dynamics, we carried out an analysis of yeast twinfilin, and we show here that twinfilin is an abundant protein that localizes to cortical actin patches in wild-type yeast cells. Native gel assays demonstrate that twinfilin binds ADP-actin monomers with higher affinity than ATP-actin monomers. A mutant twinfilin that does not interact with actin monomers in vitro no longer localizes to cortical actin patches when expressed in yeast, suggesting that the ability to interact with actin monomers may be essential for the localization of twinfilin. The localization of twinfilin to the cortical actin cytoskeleton is also disrupted in yeast strains where either the CAP1 or CAP2 gene, encoding for the alpha and beta subunits of capping protein, is deleted. Purified twinfilin and capping protein form a complex on native gels. Twinfilin also interacts with phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2), and its actin monomer-sequestering activity is inhibited by PI(4,5)P2. Based on these results, we propose a model for the biological role of twinfilin as a protein that localizes actin monomers to the sites of rapid filament assembly in cells.

Show MeSH
Twinfilin is an abundant protein in yeast cells. Yeast cell extracts as well as known concentrations of actin, cofilin, and twinfilin were run on 12% polyacrylamide gels and the proteins were subsequently visualized by Western blotting. By comparing the intensities of the protein bands in the cell extracts with the purified protein samples we estimated that the actin/cofilin/twinfilin ratio in yeast cells is ∼10:2.5:1.
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fig2: Twinfilin is an abundant protein in yeast cells. Yeast cell extracts as well as known concentrations of actin, cofilin, and twinfilin were run on 12% polyacrylamide gels and the proteins were subsequently visualized by Western blotting. By comparing the intensities of the protein bands in the cell extracts with the purified protein samples we estimated that the actin/cofilin/twinfilin ratio in yeast cells is ∼10:2.5:1.

Mentions: To understand the role of twinfilin for actin filament turnover in vivo, it is important to know the abundance of twinfilin in cells; therefore, we raised polyclonal antibodies against purified yeast twinfilin. On Western blots, the affinity-purified antibodies recognize one polypeptide with an identical mobility to that of recombinant twinfilin, indicating that these antibodies are specific and that twinfilin in yeast does not have any major posttranslational modifications. A Western blot analysis with three known concentrations of purified actin, cofilin, and twinfilin, as well as three different dilutions of wild-type yeast extracts, showed that the actin:cofilin:twinfilin ratio in these yeast extracts is ∼10:2.5:1 (Fig. 2). This shows that twinfilin is an abundant protein in yeast cells.


Interactions with PIP2, ADP-actin monomers, and capping protein regulate the activity and localization of yeast twinfilin.

Palmgren S, Ojala PJ, Wear MA, Cooper JA, Lappalainen P - J. Cell Biol. (2001)

Twinfilin is an abundant protein in yeast cells. Yeast cell extracts as well as known concentrations of actin, cofilin, and twinfilin were run on 12% polyacrylamide gels and the proteins were subsequently visualized by Western blotting. By comparing the intensities of the protein bands in the cell extracts with the purified protein samples we estimated that the actin/cofilin/twinfilin ratio in yeast cells is ∼10:2.5:1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198831&req=5

fig2: Twinfilin is an abundant protein in yeast cells. Yeast cell extracts as well as known concentrations of actin, cofilin, and twinfilin were run on 12% polyacrylamide gels and the proteins were subsequently visualized by Western blotting. By comparing the intensities of the protein bands in the cell extracts with the purified protein samples we estimated that the actin/cofilin/twinfilin ratio in yeast cells is ∼10:2.5:1.
Mentions: To understand the role of twinfilin for actin filament turnover in vivo, it is important to know the abundance of twinfilin in cells; therefore, we raised polyclonal antibodies against purified yeast twinfilin. On Western blots, the affinity-purified antibodies recognize one polypeptide with an identical mobility to that of recombinant twinfilin, indicating that these antibodies are specific and that twinfilin in yeast does not have any major posttranslational modifications. A Western blot analysis with three known concentrations of purified actin, cofilin, and twinfilin, as well as three different dilutions of wild-type yeast extracts, showed that the actin:cofilin:twinfilin ratio in these yeast extracts is ∼10:2.5:1 (Fig. 2). This shows that twinfilin is an abundant protein in yeast cells.

Bottom Line: A mutant twinfilin that does not interact with actin monomers in vitro no longer localizes to cortical actin patches when expressed in yeast, suggesting that the ability to interact with actin monomers may be essential for the localization of twinfilin.Twinfilin also interacts with phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2), and its actin monomer-sequestering activity is inhibited by PI(4,5)P2.Based on these results, we propose a model for the biological role of twinfilin as a protein that localizes actin monomers to the sites of rapid filament assembly in cells.

View Article: PubMed Central - PubMed

Affiliation: Program in Cellular Biotechnology, Institute of Biotechnology, FIN-00014 University of Helsinki, Helsinki, Finland.

ABSTRACT
Twinfilin is a ubiquitous actin monomer-binding protein that regulates actin filament turnover in yeast and mammalian cells. To elucidate the mechanism by which twinfilin contributes to actin filament dynamics, we carried out an analysis of yeast twinfilin, and we show here that twinfilin is an abundant protein that localizes to cortical actin patches in wild-type yeast cells. Native gel assays demonstrate that twinfilin binds ADP-actin monomers with higher affinity than ATP-actin monomers. A mutant twinfilin that does not interact with actin monomers in vitro no longer localizes to cortical actin patches when expressed in yeast, suggesting that the ability to interact with actin monomers may be essential for the localization of twinfilin. The localization of twinfilin to the cortical actin cytoskeleton is also disrupted in yeast strains where either the CAP1 or CAP2 gene, encoding for the alpha and beta subunits of capping protein, is deleted. Purified twinfilin and capping protein form a complex on native gels. Twinfilin also interacts with phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2), and its actin monomer-sequestering activity is inhibited by PI(4,5)P2. Based on these results, we propose a model for the biological role of twinfilin as a protein that localizes actin monomers to the sites of rapid filament assembly in cells.

Show MeSH