FUNCTIONAL ANALYSIS OF THE EH DOMAIN PROTEIN, PAN1, IN THE YEAST SACCHAROMYCES CEREVISIAE

Abstract

The actin cytoskeleton is involved in numerous fundamental processes that are essential for the development of eukaryotes. In the yeast Saccharomyces cerevisiae, a functional actin cytoskeleton is required for the maintenance of cell shape, polarized secretion, morphogenesis, endocytosis and a variety of other cellular processes. The yeast actin cytoskeleton undergoes dramatic reorganization in accordance with cell cycle progression. This reorganization has recently been shown to be under the control of the cell cycle regulator, Cdc28p kinase. In order to study this control mechanism, the isolation of rapid death mutants in the background of the START-deficient cdc28-4 mutation was attempted. The essential yeast gene PAN1, previously thought to encode a poly(A) nuclease, was isolated in this screen. PAN1, encoding an EH domain-containing protein, is identified here as a new factor required for actin cytoskeleton organization and endocytosis. Analysis of the temperature-sensitive pan1-4 mutant revealed various phenotypes associated with actin cytoskeleton disorder. Such phenotypes include abnormal bud growth, improper nuclear segregation, and defective F-actin and chitin distribution. Overexpression of the PAN1 gene also disrupted the proper organization of the actin cytoskeleton. In addition, pan1-4 displayed synthetic lethality with a null mutation of SLA1, a gene involved in membrane cytoskeleton assembly. Finally, Pan1p was found to colocalize with the cortical actin patches, suggesting a direct role for Pan1p in actin cytoskeleton organization. The role of Pan1p in endocytosis is revealed when END3, a gene encoding another EH domain protein required for endocytosis, was isolated as a multicopy suppressor of the pan1-4 mutation. Immunoprecipitation and two-hybrid protein interaction experiments indicated that Pan1p and End3p associate with each other. In the end3 null mutant, the colocalization between Pan1p and the cortical actin structures was severely affected, suggesting that End3p is required for the stable association between Pan1p and the cortical actin cytoskeleton. The physiological relevance of the association between Pan1p and End3p was further demonstrated when the pan1-4 mutant was found to be defective in both receptor-mediated and fluid-phase endocytosis. Taken together, these results provide strong evidence to indicate that the EH domain-containing protein, Pan1p, plays an essential role in actin cytoskeleton organization and endocytosis in the yeast S. cerevisiae.