Maintaining a high concentration of K+ and a low concentration of Na+ in the cytosol is important for all cells. The regulatory pathways controlling intracellular K+ and Na+ homeostasis are poorly understood in higher eukaryotic organisms. The proposed research will dissect the signaling cascades controlling K+ and Na+ homeostasis in the model multicellular organism Arabidopsis thaliana. Recently, through the identification of Arabidopsis mutants that are salt overly sensitive (sos) and the cloning and characterization of the SOS genes, we have discovered a regulatory pathway that mediates ion homeostasis in Arabidopsis. In this pathway, a myristoylated calcium-binding protein, SOS3, senses cytosolic calcium changes elicited by exposure to high Na+. SOS3 with bound calcium physically interacts with and activates the protein kinase, SOS2. The SOS3-SOS2 kinase complex phosphorylates and activates the transport activity of the plasma membrane Na+/H+ exchanger encoded by the SOS1 gene. In addition to its transport function, preliminary results show that SOS1 has a very novel protein kinase domain in its unusually long cytoplasmic tail, suggesting that it may also have a regulatory role, and could even be a novel sensor for Na+. We will use a variety of biochemical and molecular approaches to characterize the kinase activity and other potential regulatory properties of SOS1. We plan to isolate additional components of the SOS regulatory pathway through protein interaction screens, and the identification of genetic suppressors and enhancers of the existing sos mutants. Further, we will investigate the mechanism of regulation by the SOS pathway of several putative new targets, which include a transcription factor and various ion transporters on the vacuolar and plasma membranes. Lastly, we have found that the SOS1 mRNA accumulates under high salt due to transcript stabilization. Experiments are proposed to identify the cellular factors that mediate this salt-induced transcript stabilization.
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