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In vitro reconstitution of cortical actin assembly sites in budding yeast.

Lechler T, Li R - J. Cell Biol. (1997)

Bottom Line: Previous analysis indicated that an activity associated with the cell cortex promotes actin polymerization in the bud.It is composed of a novel protein, Pca1.Sequence analysis suggests that Pca1 has the potential to interact with SH3 domain-containing proteins and phospholipids.

View Article: PubMed Central - PubMed

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

ABSTRACT
We have developed a biochemical approach for identifying the components of cortical actin assembly sites in polarized yeast cells, based on a permeabilized cell assay that we established for actin assembly in vitro. Previous analysis indicated that an activity associated with the cell cortex promotes actin polymerization in the bud. After inactivation by a chemical treatment, this activity can be reconstituted back to the permeabilized cells from a cytoplasmic extract. Fractionation of the extract revealed that the reconstitution depends on two sequentially acting protein factors. Bee1, a cortical actin cytoskeletal protein with sequence homology to Wiskott-Aldrich syndrome protein, is required for the first step of the reconstitution. This finding, together with the severe defects in actin organization associated with the bee1 mutation, indicates that Bee1 protein plays a direct role in controlling actin polymerization at the cell cortex. The factor that acts in the second step of the reconstitution has been identified by conventional chromatography. It is composed of a novel protein, Pca1. Sequence analysis suggests that Pca1 has the potential to interact with SH3 domain-containing proteins and phospholipids.

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Sequential activation of actin assembly sites by ACF1 and ACF2. (A) An extract was fractionated over a Q-Sepharose column  to yield the flow through and the 0.5 M KCl eluate, which were subsequently desalted and concentrated. The urea-treated, permeabilized cells were incubated with the flow through (a), the eluate (b), or a 1:1 mixture of the two fractions (c) before Rd-actin polymerization. Rhodamine fluorescence images of representative groups of cells are shown. (B) The complementing factors in the flow through  and in the eluate are designated ACF1 and ACF2, respectively. The histograms show the percentages of the urea-treated cells that preferentially incorporated Rd-actin into the bud after the cells were incubated with the buffer, a mixture of ACF1 and ACF2, ACF1 first  and then ACF2, or ACF2 first and then ACF1. In the latter two treatments, the cells were washed with the buffer after the incubation  with the first factor. The percentages shown are averages of the results from two experiments, and the error bars are standard deviations.  Bar, 10 μm.
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Figure 3: Sequential activation of actin assembly sites by ACF1 and ACF2. (A) An extract was fractionated over a Q-Sepharose column to yield the flow through and the 0.5 M KCl eluate, which were subsequently desalted and concentrated. The urea-treated, permeabilized cells were incubated with the flow through (a), the eluate (b), or a 1:1 mixture of the two fractions (c) before Rd-actin polymerization. Rhodamine fluorescence images of representative groups of cells are shown. (B) The complementing factors in the flow through and in the eluate are designated ACF1 and ACF2, respectively. The histograms show the percentages of the urea-treated cells that preferentially incorporated Rd-actin into the bud after the cells were incubated with the buffer, a mixture of ACF1 and ACF2, ACF1 first and then ACF2, or ACF2 first and then ACF1. In the latter two treatments, the cells were washed with the buffer after the incubation with the first factor. The percentages shown are averages of the results from two experiments, and the error bars are standard deviations. Bar, 10 μm.

Mentions: The preparation of small-budded permeabilized cells and rhodamine labeling of rabbit muscle actin were carried out as described (Li et al., 1995). After the permeabilization by saponin, the cells were washed once with UB plus PI. The urea or high salt treatment was carried out by incubating the permeabilized cells in UB plus PI containing 2 M urea (Sigma Ultra grade; Sigma Chemical Co., St. Louis, MO) or 0.5 M KCl for 30 min at 27°C. The urea or high salt treated cells were washed twice with UB plus PI and then incubated with 20 μl extract for 30 min at 27°C. When ACF1 and ACF2 (for definitions see Fig. 3 legend) were added sequentially, the cells were first incubated with 10 μl of an ACF1-containing fraction for 20 min at 27°C, washed with UB plus PI, and then incubated with 10 μl of an ACF2-containing fraction for 30 min at 27°C. The cells were washed with UB plus PI, and the Rd-actin assembly reaction was carried out for 8 min as described (Li et al., 1995).


In vitro reconstitution of cortical actin assembly sites in budding yeast.

Lechler T, Li R - J. Cell Biol. (1997)

Sequential activation of actin assembly sites by ACF1 and ACF2. (A) An extract was fractionated over a Q-Sepharose column  to yield the flow through and the 0.5 M KCl eluate, which were subsequently desalted and concentrated. The urea-treated, permeabilized cells were incubated with the flow through (a), the eluate (b), or a 1:1 mixture of the two fractions (c) before Rd-actin polymerization. Rhodamine fluorescence images of representative groups of cells are shown. (B) The complementing factors in the flow through  and in the eluate are designated ACF1 and ACF2, respectively. The histograms show the percentages of the urea-treated cells that preferentially incorporated Rd-actin into the bud after the cells were incubated with the buffer, a mixture of ACF1 and ACF2, ACF1 first  and then ACF2, or ACF2 first and then ACF1. In the latter two treatments, the cells were washed with the buffer after the incubation  with the first factor. The percentages shown are averages of the results from two experiments, and the error bars are standard deviations.  Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 3: Sequential activation of actin assembly sites by ACF1 and ACF2. (A) An extract was fractionated over a Q-Sepharose column to yield the flow through and the 0.5 M KCl eluate, which were subsequently desalted and concentrated. The urea-treated, permeabilized cells were incubated with the flow through (a), the eluate (b), or a 1:1 mixture of the two fractions (c) before Rd-actin polymerization. Rhodamine fluorescence images of representative groups of cells are shown. (B) The complementing factors in the flow through and in the eluate are designated ACF1 and ACF2, respectively. The histograms show the percentages of the urea-treated cells that preferentially incorporated Rd-actin into the bud after the cells were incubated with the buffer, a mixture of ACF1 and ACF2, ACF1 first and then ACF2, or ACF2 first and then ACF1. In the latter two treatments, the cells were washed with the buffer after the incubation with the first factor. The percentages shown are averages of the results from two experiments, and the error bars are standard deviations. Bar, 10 μm.
Mentions: The preparation of small-budded permeabilized cells and rhodamine labeling of rabbit muscle actin were carried out as described (Li et al., 1995). After the permeabilization by saponin, the cells were washed once with UB plus PI. The urea or high salt treatment was carried out by incubating the permeabilized cells in UB plus PI containing 2 M urea (Sigma Ultra grade; Sigma Chemical Co., St. Louis, MO) or 0.5 M KCl for 30 min at 27°C. The urea or high salt treated cells were washed twice with UB plus PI and then incubated with 20 μl extract for 30 min at 27°C. When ACF1 and ACF2 (for definitions see Fig. 3 legend) were added sequentially, the cells were first incubated with 10 μl of an ACF1-containing fraction for 20 min at 27°C, washed with UB plus PI, and then incubated with 10 μl of an ACF2-containing fraction for 30 min at 27°C. The cells were washed with UB plus PI, and the Rd-actin assembly reaction was carried out for 8 min as described (Li et al., 1995).

Bottom Line: Previous analysis indicated that an activity associated with the cell cortex promotes actin polymerization in the bud.It is composed of a novel protein, Pca1.Sequence analysis suggests that Pca1 has the potential to interact with SH3 domain-containing proteins and phospholipids.

View Article: PubMed Central - PubMed

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

ABSTRACT
We have developed a biochemical approach for identifying the components of cortical actin assembly sites in polarized yeast cells, based on a permeabilized cell assay that we established for actin assembly in vitro. Previous analysis indicated that an activity associated with the cell cortex promotes actin polymerization in the bud. After inactivation by a chemical treatment, this activity can be reconstituted back to the permeabilized cells from a cytoplasmic extract. Fractionation of the extract revealed that the reconstitution depends on two sequentially acting protein factors. Bee1, a cortical actin cytoskeletal protein with sequence homology to Wiskott-Aldrich syndrome protein, is required for the first step of the reconstitution. This finding, together with the severe defects in actin organization associated with the bee1 mutation, indicates that Bee1 protein plays a direct role in controlling actin polymerization at the cell cortex. The factor that acts in the second step of the reconstitution has been identified by conventional chromatography. It is composed of a novel protein, Pca1. Sequence analysis suggests that Pca1 has the potential to interact with SH3 domain-containing proteins and phospholipids.

Show MeSH
Related in: MedlinePlus