In vitro reconstitution of cortical actin assembly sites in budding yeast

Terry Lechler, Rong Li

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish (US)
Pages (from-to)95-103
Number of pages9
JournalJournal of Cell Biology
Volume138
Issue number1
DOIs
StatePublished - Jul 14 1997

ASJC Scopus subject areas

  • Cell Biology

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