A novel member of the rho family of small GTP-binding proteins is specifically required for cytokinesis.

Abstract

Abstract. Several members of the rho/rac family of small GTP-binding proteins are known to regulate the distribution of the actin cytoskeleton in various subcellular processes. We describe here a novel rac protein, racE, which is specifically required for cytokinesis, an actomyosin-mediated process. The racE gene was isolated in a molecular genetic screen devised to isolate genes required for cytokinesis in Dictyostelium. Phenotypic characterization of racE mutants revealed that racE is not essential for any other cell motility event, including phagocytosis, chemotaxis, capping, or development. Our data provide the first genetic evidence for the essential requirement of a rho-like protein, specifically in cytokinesis, and suggest a role for these proteins in coordinating cytokinesis with the mitotic events of the cell cycle.T HE intimate association between mitosis and cytokinesis requires a means of coordination between these two processes to insure that the newly duplicated nuclei segregate properly with half of the cytoplasm into the daughter cells. Although much is known about these processes, the mechanism(s) by which they are coordinated remains unknown. The regulation of the mitotic cell cycle has been intensively studied over the last several years. Biochemical and genetic approaches have combined to identify many of the key proteins that control different aspects of the cell cycle. In addition, many of the structural proteins that compose the mitotic apparatus have been characterized. Similarly, much is understood about how cells achieve proper cytoplasmic division. In animal cells, this involves the formation of an equatorial contractile ring that consists largely of actin and myosin and constricts to divide the cell into two . However, it is not understood how these proteins localize to the equator of the cell at the appropriate time and in the correct orientation. From the work of Rappaport (1990), it is clear that the astral microtubules of the mitotic apparatus are intimately involved in determining the placement of the contractile ring. What is not clear is what kind of signals may be involved or how they may be transmitted by the mitotic apparatus to the cell cortex.The rho family of ras-related small GTP-binding proteins (including rho, cdc42, and rac proteins) are known to have profound effects on the actin cytoskeleton (Hall, 1994). Rho proteins have been implicated in the regulation of cytokinesis in both sand dollar (Mabuchi et al., 1993)

DGAP1, a homologue of rasGTPase activating proteins that controls growth, cytokinesis, and development in Dictyostelium discoideum

“…(2) Mutants altered in proteins that play a regulatory role in mitosis. Coronin, a WD40-repeat protein involved in dynamic rearrangements of the actin system [23], racE [24], and DGAP1 belong to this category. These and certainly more regulatory proteins are required in D. discoideum cells to guarantee the coordination in time and space of cytoskeletal activities in the cell cortex.…”

Role of Dictyostelium racE in cytokinesis: mutational analysis and localization studies by use of green fluorescent protein.

“…The distribution of GFP-N25racE in these cells remained unchanged, localizing primarily to the plasma membrane in both interphase and dividing cells ( Figure 5, B, C, E, and F). Localization of GFP-racE Is Mediated by the C Terminus of racE Although all members of the rho family of small GTPases are very similar to each other, a salient feature of racE is the unique sequence near its carboxyl terminus (Larochelle et al, 1996). In comparison to all other members of this family, the C-terminal domain of racE is extended by about 28 amino acids.…”

“…The active mutant is the result of reduced GTPase activity (Garrett et al, 1989) and the inactive mutant is the result of an inability to exchange GTP for bound GDP (Feig and Cooper, 1988). A fusion protein was also designed, using PCR, to replace the short hypervariable carboxyl-terminal region of racC with the extended carboxyl-terminal region of racE (Larochelle et al, 1996). This protein, named racC/E tail, contained amino acid residues 1-180 of racC and residues 184-223 of racE.…”

“…The Function of racE Cannot Be Replaced by a Closely Related Protein Currently, the protein most similar to Dictyostelium racE in the GenBank database is the Dictyostelium racC protein (Bush et al, 1993;Larochelle et al, 1996). Although racC shares 54% identical residues (68% overall conserved) with racE, it apparently cannot substi- tute for racE in cytokinesis since racC is expressed in racE null cells.…”

“…Recently, using a genetic screen for Dictyostelium cytokinesis mutants, we identified a novel member of the rho family of GTPases, which we designated as racE (Larochelle et al, 1996). This protein appears to have a specific role during cytokinesis since a null mutation in the racE gene does not perturb any cellular event except for cell division.…”