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Calmodulin Kinase 1 Regulates the Vacuolar Calcium Exporter Yvc1p in Saccharomyces cerevisiae

Jennifer L Kepler, Zinayida Nektalova, Jennifer Muir and Pamela A Marshall

We are interested in analyzing the key proteins involved in calcium homeostasis in Saccharomyces cerevisiae and undertook deletion mutant analysis, assaying cytosolic calcium concentration after a single extracellular pulse of 100 mM. In wild type cells a spike in cytosolic calcium was observed, followed by a decrease to approximately pre-pulse baseline level, which was maintained for the remainder of the 13 minute assay. In yeast mutants cmd1- 6, Δcnb1, Δcrz1, Δcmk1, and Δcmk2, an initial spike was also seen, followed by the baseline return, and then a second rise in cytosolic calcium after six minutes was observed. We wanted to identify the origin of this second rise in cytosolic calcium in the deletion mutants, hypothesizing it was due to the vacuolar calcium exporter channel, Yvc1p, aberrantly responding to the hyperosmolarity of the cytosol. In Δcmk1Δyvc1 cells, the second rise was absent. In Δcnb1Δyvc1 cells, the second rise was still observed and was not due to Vcx1p activity. We propose that in the presence of high extracellular calcium, an initial feedback mechanism regulated by calcineurin, in response to an elevated level of intracellular calcium entry, inhibits the plasma membrane calcium channel, while calmodulin kinase 1 inhibits Yvc1p during an additional feedback response.