RacF1, a novel member of the Rho protein family in Dictyostelium discoideum, associates transiently with cell contact areas, macropinosomes, and phagosomes

Using a PCR approach we have isolated racF1, a novel member of the Rho fami ly in Dictyostelium. The racF1 gene encodes a protein of 193 amino acids an d is constitutively expressed throughout the Dictyostelium life cycle. High est identity (94%) was found to a RacF2 isoform, to Dictyostelium Rac1A, Ra c1B, and Rac1C (70%), and to Rac proteins of animal species (64 - 69%). To investigate the role of RacF1 in cyto skeleton-dependent processes, we have fused it at its amino-terminus with green fluorescent protein (GFP) and st udied the dynamics of subcellular redistribution using a confocal laser sca nning microscope and a double-view microscope system. GFP-RacF1 was homogen eously distributed in the cytosol and accumulated at the plasma membrane, e specially at regions of transient intercellular contacts. GFP-RacF1 also lo calized transiently to macropinosomes and phagocytic cups and was gradually released within <1 min after formation of the endocytic vesicle or the pha gosome, respectively. On stimulation with cAMP, no enrichment of GFP-RacF1 was observed in leading fronts, from which it was found to be initially exc luded. Cell lines were obtained using homologous recombination that express ed a truncated racF1 gene lacking sequences encoding the carboxyl-terminal region responsible for membrane targeting. These cells displayed normal pha gocytosis, endocytosis, and exocytosis rates. Our results suggest that RacF 1 associates with dynamic structures that are formed during pinocytosis and phagocytosis. Although RacF1 appears not to be essential, it might act in concert and/or share functions with other members of the Rho family in the regulation of a subset of cytoskeletal rearrangements that are required for these processes.