UBC Theses and Dissertations
Analysis of the effects of the Rap1 and Rasg genes on dictyostelium discoideum cell morphology Rebstein, Patrick James
The rapl gene of Dictyostelium discoideum is a member of the ras-gene superfamily of low molecular weight GTPase proteins. The D. discoideum rapl gene is expressed both during growth and development. To further characterize rapl, the sequence and organization of the genomic DNA encoding the rapl gene and 1 kbp of the 5' flanking region was determined. The 5' flanking region could activate expression of the (3-galactosidase reporter gene upon transformation into D. discoideum. To examine the action of the Rapl protein in D. discoideum, the rapl cDNA was expressed under the control of the inducible discoidin promoter. Overexpression of the Rapl protein correlated with the appearance of morphologically aberrant vegetative amebae: cells were extensively spread and flattened with an increase in F-actin staining around the cell periphery. Expression of the rapl cDNA also prevented cell detachment from the substratum upon treatment with azide. When starved D. discoideum amebae are stimulated with HL5 medium, the cells rapidly respond by rounding up. By contrast, the Rapl transformant cells showed a pronounced delay in rounding. Site directed mutagenesis was used to determine the requirements for specific conserved amino acids for these effects caused by overexpression of Rapl. The substitution G10V, predicted to prevent nucleotide binding, and the substitution S17N, predicted to restrict the protein to the GDP-bound state, both abolished the ability of Rapl to affect cell morphology, suggesting that GTP binding is required for Rapl activity. Moreover, the substitution G12V which is predicted to increase the proportion of protein binding GTP, modestly enhanced the ability of Rapl to inhibit the rounding of starved cells after nutrient stimulation. By contrast, substitutions at amino acid 38 in the presumptive effector domain reduced but did not totally abolish the ability of Rapl to affect cell morphology. The substitution T61Q, which impairs the ability of mammalian Rapl to revert the phenotype of Ras transformed mammalian cells, did not alter the effect of Rapl on D. discoideum cell morphology. Thus, although the effects of Rapl on D. discoideum cell morphology appear to be regulated by GTP, the unexpected effects of substitutions at amino acid positions 38 and 61 suggests that the Rapl induced effects may involve different effector and regulatory molecules from that required to revert the phenotype of Ras transformed cells. A D. discoideum transformant expressing activated RasG-G12T protein (RasG-G12T) had an altered cell morphology similar to that of Rapl overexpression: the cells became flattened and spread with a concomitant distribution of F-actin around the cell periphery. RasG-G12T cells also failed to round up and detach upon exposure to azide. However, expression of activated RasG-G12T resulted in the formation of multinucleate cells whereas Rapl expressing transformants remained mononucleate.
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