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Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115
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 null 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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