Abstract #66

RHO5 is necessary for maintenance of redox homeostasis in yeast. Komudi Singh, Pil Jung Kang, Hay-Oak Park. Molecular Genetics, The Ohio State University, Columbus, OH.
   Several previous studies have suggested a link between GTPases and production of reactive oxygen species (ROS) or oxidant-mediated signaling in various cells. Changes in the intracellular redox state appear to regulate several critical intracellular pathways in mammalian cells. In yeast, as in higher eukaryotes, ROS are produced as normal by-products of cellular metabolism. The redox balance is disturbed when yeast cells are exposed to diverse environmental stress conditions, such as the presence of oxidants, heat shock, or metal ions. Yeast cells have evolved defense systems to avoid the deleterious consequence of ROS accumulation. However, the signaling pathways involved in the oxidative stress response or maintenance of redox homeostasis are not well established. Here, we show that Rho5 GTPase is necessary for maintenance of redox homeostasis in budding yeast. The rho5 deletion and dominant negative rho5(K16N) mutants were resistant to oxidants such as Paraquat, a superoxide generating agent, and DEM (diethyl maleate), a thiol-specific oxidant that depletes glutathione in the cell. In contrast, the dominant activating rho5(G12V) mutant was hypersensitive to these oxidants. Analysis of these cells by fluorescence microscopy and flow cytometry after staining with an oxidation-sensitive dye indicated that the rho5(G12V) mutant cells accumulated a high level of ROS upon exposure to the oxidants such as hydrogen peroxide or DEM. Interestingly, a recent high-throughput proteomics study indicated that Rho5 co-purified with Trr1 (thioredoxin reductase) (Ho et al., 2002), a key component in regulation of redox homeostasis. Thioredoxin reductase reduces the oxidized disulfide form of thioredoxins using NADPH, and is required for protection of yeast cells against both oxidative and reductive stress (Trotter & Grant, 2002). We found that Rho5 interacted with Trr1 in a guanine nucleotide-dependent manner by two-hybrid and in vitro assays. Taken together, these data suggest that Rho5 is involved in redox homeostasis in yeast by regulating thioredoxin reductase.

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