6 pages, 6 figures, 29 references.The Arabidopsis thaliana SOS1 protein is a putative Na H antiporter that functions in Na extrusion and is essential for the NaCl tolerance of plants. sos1 mutant plants share phenotypic similarities with mutants lacking the protein kinase SOS2 and the Ca2 sensor SOS3. To investigate whether the three SOS proteins function in the same response pathway, we have reconstituted the SOS system in yeast cells. Expression of SOS1 improved the Na tolerance of yeast mutants lacking endogenous Na transporters. Coexpression of SOS2 and SOS3 dramatically increased SOS1- dependent Na tolerance, whereas SOS2 or SOS3 individually had no effect. The SOS2 SOS3 kinase complex promoted the phosphorylation of SOS1. A constitutively active form of SOS2 phosphorylated SOS1 in vitro independently of SOS3, but could not fully substitute for the SOS2 SOS3 kinase complex for activation of SOS1 in vivo. Further, we show that SOS3 recruits SOS2 to the plasma membrane. Although sos1 mutant plants display defective K uptake at low external concentrations, neither the unmodified nor the SOS2 SOS3-activated SOS1 protein showed K transport capacity in vivo, suggesting that the role of SOS1 on K uptake is indirect. Our results provide an example of functional reconstitution of a plant response pathway in a heterologous system and demonstrate that the SOS1 ion transporter, the SOS2 protein kinase, and its associated Ca2 sensor SOS3 constitute a functional module. We propose a model in which SOS3 activates and directs SOS2 to the plasma membrane for the stimulatory phosphorylation of the Na transporter SOS1.This work was supported by Spanish Ministerio de Ciencia y Tecnología Grant BIO2000-0938 (to F.J.Q. and J.M.P.) and by National Institutes of Health Grant R01GM59138 (to J.-K.Z.).Peer reviewed