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Identification of the domains of RasG-G12T that are required to produce defects in aggregation and cytoskeletal function in dictyostelium discoideum

University of British Columbia

Dictyostelium transformants expressing an activated rasG gene, rasG-G12T, do not aggregate when starved for nutrients. In addition, expression of the activated RasG protein results in vegetative cell populations with heterogeneous morphology. Some cells are extensively flattened and spread and exhibit lateral or dorsal membrane ruffling while others which are less flattened, exhibit prominent dorsal membrane ruffling. The expression of RasG-G12T also causes a slight increase in the average number of nuclei and results in a redistribution of F-actin to the cell periphery. These results suggest that RasG has roles in both cytoskeletal and developmental regulation in Dictyostelium. To identify the functional residues required for the downstream effects of activated RasG, amino acid substitutions have been introduced into RasG-G12T within the effector domain (Tyr32-Tyr40) or within the effector distal flanking domain. Cells expressing RasG-G12T with amino acid substitutions in the effector domain (T35S or Y40C) showed normal morphology. These cells also aggregated and differentiated normally, suggesting that the defects cause by RasG-G12T required interaction of the effector domain with a downstream protein(s). In contrast an amino acid substitution in the effector distal flanking domain (T45Q) prevented the RasG-G12T induced block in aggregation, but was not able to prevent the cytoskeletal defect. This result suggests that the cytoskeletal and developmental defects induced by RasG-G12T result from the interaction of the protein with different downstream effector molecules.

Thesis/Dissertation

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2009-04-30

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http://hdl.handle.net/2429/7762

Microbiology and Immunology

University of British Columbia

UBCV

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