Calcium serves as a second messenger for signal transduction in nearly all eukaryotic cells. While much is known about the mechanisms which control and respond to Ca2+ signals, many important components of this process have not been identified. For example, Ca2+ signaling is necessary for the normal response of human T-cells to antigens, yet the critical Ca2+ channels in the plasma membrane have not been identified and the events downstream of calcineurin, a Ca2+-dependent protein phosphatase, have not been completely characterized. Our preliminary results reveal that the budding yeast Saccharomyces cerevisiae also produces Ca2+ signals and may do so using conserved mechanisms. The most significant conclusion from our studies is that, for the first time, the mechanism of Ca2+ signaling in yeast can be dissected using genetic approaches. The long-term objective of this proposal is to take advantage of the experimental strengths of the system and produce a detailed molecular model of Ca2+ signaling and the related process of Ca2+ homeostasis. Our preliminary work has revealed two new enzymes that help control cytosolic Ca2+, a high-affinity Ca2+ ATPase encoded by PMC1 and a low- affinity H+/Ca2+ exchanger encoded by VCX1. Genetic studies show that these Ca2+ transporters not only control the activation of calcineurin by calmodulin, but are themselves feedback regulated by calcineurin. The exchanger appears to be directly or indirectly inactivated by calcineurin whereas PMCI gene expression appears to be markedly induced by calcineurin. This proposal aims to define how calcineurin accomplishes these specific effects. We will clone and characterize the regulatory factors downstream of calcineurin, organize them into a functional sequence, determine how these factors affect the targets and how they respond to calcineurin. The results obtained from these studies will be interesting to compare with the information emerging from studies of Ca2+ signaling in mammalian cells. We also present new evidence that Ca2+ signaling in yeast is physiologically important. We observed two new environmental conditions which activate calcineurin by evoking putative Ca2+ signals. The Ca2+ channels and regulatory factors predicted by these findings are currently unknown. Accordingly, we propose resembling capacitative Ca2+ entry in mammalian cells is described. We will perform genetic screens that should reveal the factors required for the increased Ca2+ influx observed as a response to depletion of intracellular Ca2+ pools. To accomplish these goals we will use specifically designed genetic, molecular, cell biological, physiological, and biochemical techniques.
