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Journal Article
Functional overlap of the dictyostelium RasG, RasD and RasB proteins
M. Khosla, G.B. Spiegelman, R. Insall, G. Weeks
Journal of Cell Science 2000 113: 1427-1434;
M. Khosla
Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.
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G.B. Spiegelman
Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.
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R. Insall
Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.
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G. Weeks
Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.
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Summary

Disruption of the rasG gene in Dictyostelium discoideum results in several distinct phenotypes: a defect in cytokinesis, reduced motility and reduced growth. Reintroduction of the rasG gene restores all of the properties of the rasG(-) cells to those of the wild type. To determine whether the defects are due to impaired interactions with a single or multiple downstream effectors, we tested the ability of the highly related but non identical Dictyostelium ras genes, rasD and rasB, to rescue the defects. Introduction of the rasD gene under the control of the rasG promoter into rasG null (rasG(-)) cells corrected all phenotypes except the motility defect, suggesting that motility is regulated by a RasG mediated pathway that is different to those regulating growth or cytokinesis. Western blot analysis of RasD protein levels revealed that vegetative rasG(-)cells contained considerably more protein than the parental AX-3 cells, suggesting that RasD protein levels are negatively regulated in vegetative cells by RasG. The level of RasD was enhanced when the rasD gene was introduced under the control of the rasG promoter, and this increase in protein is presumably responsible for the reversal of the growth and cytokinesis defects of the rasG(-)cells. Thus, RasD protein levels are controlled by the level of RasG, but not by the level of RasD. Introduction of the rasB gene under the control of the rasG promoter into rasG(-) cells produced a complex phenotype. The transformants were extremely small and mononucleate and exhibited enhanced motility. However, the growth of these cells was considerably slower than the growth of the rasG(-) cells, suggesting the possibility that high levels of RasB inhibit an essential process. This was confirmed by expressing rasB in wild-type cells; the resulting transformants exhibited severely impaired growth. When RasB protein levels were determined by western blot analysis, it was found that levels were higher in the rasG(-)cells than they were in the wild-type parental, suggesting that RasG also negatively regulates rasB expression in vegetative cells. Overexpression of rasB in the rasG(-)cells also reduced the level of RasD protein. In view of the fact that alternate Ras proteins correct some, but not all, of the defects exhibited by the rasG(-) cells, we propose that RasG interacts with more than one downstream effector. In addition, it is clear that the levels of the various Ras proteins are tightly regulated in vegetative cells and that overexpression can be deleterious.

  • © 2000 by Company of Biologists
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Journal Article
Functional overlap of the dictyostelium RasG, RasD and RasB proteins
M. Khosla, G.B. Spiegelman, R. Insall, G. Weeks
Journal of Cell Science 2000 113: 1427-1434;
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Journal Article
Functional overlap of the dictyostelium RasG, RasD and RasB proteins
M. Khosla, G.B. Spiegelman, R. Insall, G. Weeks
Journal of Cell Science 2000 113: 1427-1434;

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