The Ca 2+ /Calcineurin-Regulated cup Gene Family in Dictyostelium discoideum and Its Possible Involvement in Development

Abstract

ABSTRACT Changes in free intracellular Ca 2+ are thought to regulate several major processes during Dictyostelium development, including cell aggregation and cell type-specific gene expression, but the mechanisms involved are unclear. To learn more about Ca 2+ signaling and Ca 2+ homeostasis in this organism, we used suppression subtractive hybridization to identify genes up-regulated by high extracellular Ca 2+ . Unexpectedly, many of the genes identified belong to a novel gene family (termed cup ) with seven members. In vegetative cells, the cup genes were up-regulated by high Ca 2+ but not by other ions or by heat, oxidative, or osmotic stress. cup induction by Ca 2+ was blocked completely by inhibitors of calcineurin and protein synthesis. In developing cells, cup expression was high during aggregation and late development but low during the slug stage. This pattern correlates closely with reported levels of free intracellular Ca 2+ during development. The cup gene products are highly homologous, acidic proteins possessing putative ricin domains. BLAST searches failed to reveal homologs in other organisms, but Western analyses suggested that Cup-like proteins might exist in certain other cellular slime mold species. Localization experiments indicated that Cup proteins are primarily cytoplasmic but become cell membrane-associated during Ca 2+ stress and cell aggregation. When cup expression was down-regulated by antisense RNA, the cells failed to aggregate. However, this developmental block was overcome by partially up-regulating cup expression. Together, these results suggest that the Cup proteins in Dictyostelium might play an important role in stabilizing and/or regulating the cell membrane during Ca 2+ stress and/or certain stages of development.