ROLE OF DICTYOSTELIUM RACE IN CYTOKINESIS - MUTATIONAL ANALYSIS AND LOCALIZATION STUDIES BY USE OF GREEN FLUORESCENT PROTEIN

The small GTPase racE is essential for cytokinesis in Dictyostelium bu t its precise role in cell division is not known. To determine the mol ecular mechanism of racE function, we undertook a mutational analysis of racE. The exogenous expression of either wild-type racE or a consti tutively active V20racE mutant effectively rescues the cytokinesis def iciency of racE null cells. In contrast, a constitutively inactive N25 racE mutant fails to rescue the cytokinesis deficiency. Thus, cytokine sis requires only the activation of racE by GTP and not the inactivati on of racE by hydrolysis of GTP. To determine the spatial distribution of racE, we created a fusion protein with GFP at the amino terminus o f racE. Remarkably, GFP-racE fusion protein was fully competent to res cue the phenotype of racE null cells and, therefore, must reside in th e same location as native racE. We found that GFP-racE localized to th e plasma membrane of the cell throughout the entire cell cycle. Furthe rmore, constitutively active and inactive GFP-racE fusion proteins als o localized to the plasma membrane. We mapped the domain required for plasma membrane localization to the carboxyl-terminal 40 amino acids o f racE. This domain, however, is not sufficient to confer racE functio n onto a closely related GTPase. Taken together, these results suggest that racE functions at the cell cortex but it is-not involved in dete rmining the timing or placement of the contractile ring.