Trix, a novel Rac guanine-nucleotide exchange factor from Dictyostelium discoideum is an actin-binding protein and accumulates at endosomes.

Small Rho family GTPases are involved in regulation of actin cytoskeleton dynamics. These molecular switches are themselves mainly controlled by specific GTPase-activating proteins (GAPs) and guanine-nucleotide exchange factors (GEFs). We have cloned and initially characterized a novel putative RhoGEF from Dictyostelium discoideum. The predicted 135-kDa protein displays a unique domain organization in its N-terminus by harboring two type3 calponin homology (CH) domains followed by a single type1 CH domain. The C-terminal region encompasses a diffuse B-cell lymphoma homology/pleckstrin homology tandem domain that is typically found in RhoGEFs. We therefore refer to this protein as Trix (triple CH-domain array exchange factor). A recombinant N-terminal region of Trix carrying all three CH domains binds to F-actin and bundles actin filaments. Trix-null mutants are viable and display only subtle defects when compared to wild-type cells with the exception of a substantial decrease in exocytosis of a fluid-phase marker. GFP fusions with the full-length protein or the N-terminal part containing all three CH domains revealed that Trix localizes to the cortical region and strongly accumulates on late endosomes. Our results suggest that Trix is specifically involved in a Rho GTPase-signaling pathway that is required for regulation of the actin cytoskeleton during exocytosis.