Yeast magnesium starvation, 90 minutes

To learn about the cellular processes involved in Mg2+ transport and the mechanisms allowing cells to cope with low Mg2+ availability, we performed RNA expression profiling experiments, and followed changes in gene activity upon Mg2+ depletion on a genome-wide scale. A striking portion of genes up-regulated under Mg2+ depletion is also induced by high Ca2+ and/or alkalinization. Among the genes significantly up-regulated by Mg2+ starvation, Ca2+ stress and alkalinization are ENA1 (encoding a P-type ATPase sodium pump) and PHO89 (encoding a sodium/phosphate cotransporter). We show that up-regulation of these genes is dependent on the calcineurin/Crz1p signaling pathway. Similarly to Ca2+ stress, Mg2+ starvation induces translocation of the transcription factor Crz1p from the cytoplasm into the nucleus. The up-regulation of ENA1 and PHO89 upon Mg2+ starvation depends on extracellular Ca2+. Using fluorescence resonance energy transfer microscopy we demonstrate that removal of Mg2+ results in an immediate increase in free cytoplasmic Ca2+. This effect is dependent on external Ca2+. Results presented indicate that Mg2+ depletion in yeast cells leads to enhanced cellular Ca2+ concentrations, which activate the Crz1p/calcineurin pathway. We provide evidence that calcineurin/Crz1p signaling is crucial for yeast cells to cope with Mg2+ depletion stress. Keywords: stress response (magnesium starvation) Mg2+ starvation and RNA isolation - For each experiment two identical yeast cultures were grown in synthetic medium containing 1 mM MgCl2 to OD600 = 0.5. The cultures were centrifuged, washed twice with pre-warmed synthetic medium containing either 1 mM MgCl2 (+Mg2+) or no MgCl2 (-Mg2+), and resuspended in the same medium. For time course experiments aliquots were removed at the indicated time points; for all other experiments cells were harvested 70 minutes after the 1st wash. For the FK506 experiment, FK506 (Fujisawa GmbH, Munich) in DMSO was added to a final concentration of 1 µg/ml (=1.25 µM) 10 minutes before centrifugation, and for all subsequent steps. Total RNA was isolated using the hot acidic phenol method (1). For microarray experiments three chloroform extractions were performed instead of one. DNA microarray analyses - Yeast DNA arrays were obtained from the Ontario Cancer Institute Microarray Centre. Reverse transcription, probe cleanup, and microarray hybridization were performed according to the manufacturer’s protocol. Two individual experiments including dye-swap were performed. Microarrays were read using an axon GenePix 4000B laser scanner (Axon Instruments) and analyzed with the GenePix Pro 3.0 software. The Saccharomyces Genome Database was used to extract the information on the genes regulated by Mg2+ starvation. The geneXplorer 2.0 : Megayeast site from Stanford University was used to search for genes induced under general stress conditions.