Ege A, a Novel C2 Domain Containing Protein, Is Essential for GPCR-Mediated Gene Expression in Dictyostelium

Abstract

During early stages of development, expression of aggregative genes in Dictyostelium is regulated by G protein-linked signaling pathways. We have isolated an aggregation-deficient mutant from a restriction enzyme-mediated insertional mutagenesis screen and have obtained its cDNA. Since the mutant expresses prestarvation genes but fails to express early genes, such as cAR1 and GP80, during development, we designated it early gene expression A ( ege A). Ege A, encoding a cytosolic protein of 26 kDa, along with Ege B, belongs to a novel C2 domain-containing gene family. While Ege A mRNA is expressed during the first 2 h of development, Ege B is expressed at later stages. Ege A is not directly required for either G protein-mediated actin polymerization or activation of adenylyl cyclase. Ege A overexpressing and ege A − cells display similar phenotypes, suggesting that an optimal level of Ege A is required for proper function. Constitutive expression of a fully functional cAR1-YFP enables ege A − cells to form loose aggregates, but cAR1-YFP/ ege A − cells are still unable to express GP80, suggesting that losses of gene expression were not solely due to a lack of cAR1. Overexpression of PKAcat, the constitutively active subunit of PKA, does not rescue the ege A − phenotype, suggesting that PKA is not located downstream from Ege A in the signaling pathway. We propose that Ege A is a novel cytosolic component required by early gene expression.