Date of Award
Doctor of Philosophy
Genetics, Development and Cell Biology
Alan M. Myers
Rho1p is a guanine nucleotide binding protein coded for by the Saccharomyces cerevisiae gene RHO1, which is homologous to p21[superscript] ras and other members of the ras superfamily of small molecular weight GTP binding proteins. This protein family is highly conserved in evolution and in some instances is known to be involved in control of cell proliferation. The precise physiological functions of most p21[superscript] ras-related proteins, however, are not known. In order to gain further insight into the physiological function of rho proteins, this research addressed the genetic and biochemical characterization of Rho1p, as well as the subcellular localization of this protein;Yeast strains were constructed to cause either high or low levels of Rho1p activity by modification of the normal RHO1 gene. Elevated levels of Rho1p caused greatly enlarged, unbudded, polyploid cells, whereas low levels caused a phenotype indicative of excessive budding. Cells expressing high Rho1p activity also displayed an abnormal actin cytoskeleton. Thus, Rho1p functions as a negative regulator of morphological development in the cell cycle, possibly involving the actin cytoskeleton;Rho1p was localized in yeast cells by immunofluorescence microscopy and subcellular fractionation using Rho1p specific antibodies. Immunofluorescence microscopy showed a punctate staining pattern around the periphery of the cell. Subcellular fractionation studies utilized immunoblots to localize Rho1p to a membrane fraction. In density gradient fractionation of the membrane fraction, Rho1p colocalized with Golgi apparatus markers. In fractionation studies using secretory mutants that accumulate secretory vesicles, there was a coincident accumulation of Rho1p. Thus, Rho1p is located in the Golgi apparatus and secretory vesicles present at the cell's periphery;Rho1p was purified and biochemically characterized regarding its guanine nucleotide binding and hydrolysis properties. Rho1p bound GTP with rates of 0.08, 0.10, and 0.034 pmol GTP bound/pmol Rho1p/min at 2 [mu]M GTP and at free magnesium levels of 0.2 [mu]M, 50 [mu]M, and 10 mM, respectively. The protein had intrinsic GTPase specific activity of 0.021, 0.032, and 0.028 pmol P[subscript] i released/pmol Rho1p/min, respectively, at these same free magnesium concentrations. This GTPase specific activity was increased by incubation with the BEM2 gene product, Bem2p, another gene known to be involved in the process of bud formation. This finding provides a biochemical link between Bem2p and Rho1p, which have previously been shown to cause similar phenotypes when they are either overexpressed (RHO1) or nonfunctioning (BEM2).
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Joni Marie Seeling Johnson
Johnson, Joni Marie Seeling, "Genetic and biochemical characterization of the p21[superscript ras]-related protein Rho1p in the yeast Saccharomyces cerevisiae " (1992). Retrospective Theses and Dissertations. 9838.