In vitro reconstitution of cortical actin assembly sites in budding yeast

Journal Article

We have developed a biochemical approach for identifying the components of cortical actin assembly sites in polarized yeast cells, based on a permeabilized celt 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 Peal has the potential to interact with SH3 domain-containing proteins and phospholipids.

Full Text

Duke Authors

Cited Authors

  • Lechler, T; Li, R

Published Date

  • 1997

Published In

Volume / Issue

  • 138 / 1

Start / End Page

  • 95 - 103

PubMed ID

  • 9214384

International Standard Serial Number (ISSN)

  • 0021-9525

Digital Object Identifier (DOI)

  • 10.1083/jcb.138.1.95