In vitro reconstitution of cortical actin assembly sites in budding yeast.
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.
Lechler
T
T
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Li
R
R
D000199:Actins / Q000378:metabolism*; D000251:Adenosine Triphosphatases; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D027682:Cation Transport Proteins; D003593:Cytoplasm / Q000378:metabolism; D003598:Cytoskeletal Proteins; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D014508:Urea / Q000494:pharmacology; D051316:Wiskott-Aldrich Syndrome Protein
D000199:Actins; D027682:Cation Transport Proteins; D003598:Cytoskeletal Proteins; D005656:Fungal Proteins; C480361:LAS17 protein, S cerevisiae; D008840:Microfilament Proteins; C093603:PCA1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D051316:Wiskott-Aldrich Syndrome Protein; D014508:Urea; D000251:Adenosine Triphosphatases