Altered morphology of vegetative amoebae induced by increased expression of the Dictyostelium discoideum ras-related gene rap1

Abstract

The rap1 gene of Dictyostelium discoideum is a member of the ras-gene superfamily of low molecular weight GTPase proteins. The rap1 gene is expressed both during growth and development in D. discoideum. To examine the action of the Rap1 protein in D. discoideum, the rap1 cDNA was expressed under the control of the inducible discoidin promoter. Treatment with conditioned media, which induces the discoidin promoter, increased Rap1 protein levels in vegetative cells approximately six fold. Overexpression of the Rap1 protein correlated with the appearance of morphologically aberrant vegetative amoebae: cells were extensively spread and flattened. The distribution of F-actin was altered in these cells, with an increase in actin staining around the cell periphery. Induction of the discoidin promoter by starvation in the rap1 transformants also resulted in spread flat cells. When starved D. discoideum amoebae are refed with HL5 media, the cells rapidly respond by rounding up. By contrast, the rap1 transformant cells showed a pronounced delay in rounding up. Rapid tyrosine phosphorylation of a p45 protein occurred in both control cells and the rap1 transformant upon refeeding, implying that the signal transduction pathway leading to tyrosine phosphorylation remained functional in the rap1 transformant. We propose that the Rap1 protein functions in the regulation of cell morphology in D. discoideum.