An Osmotically Induced Cytosolic Ca2+ Transient Activates Calcineurin Signaling to Mediate Ion Homeostasis and Salt Tolerance of Saccharomyces cerevisiae

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212

Exposure of the yeast Saccharomyces cerevisiae to alkaline stress resulted in adaptive changes that involved remodeling the gene expression. Recent evidence suggested that the calcium-activated protein phosphatase calcineurin could play a role in alkaline stress signaling. By using an aequorin luminescence reporter, we showed that alkaline stress resulted in a sharp and transient rise in cytoplasmic calcium. This increase was largely abolished by addition of EGTA to the medium or in cells lacking Mid1 or Cch1, components of the high affinity cell membrane calcium channel. Under these circumstances, the alkaline response of different calcineurin-sensitive transcriptional promoters was also blocked. Therefore, exposure to alkali resulted in entry of calcium from the external medium, and this triggered a calcineurin-mediated response. The involvement of calcineurin and Crz1/Tcn1, the transcription factor activated by the phosphatase, in the transcriptional response triggered by alkalinization has been globally assessed by DNA microarray analysis in a time course experiment using calcineurin-deficient (cnb1) and crz1 mutants. We found that exposure to pH 8.0 increased at least 2-fold the mRNA levels of 266 genes. In many cases (60%) the response was rather early (peak after 10 min). The transcriptional response of 27 induced genes (10%) was reduced or fully abolished in cnb1 cells. In general, the response of crz1 mutants was similar to that of calcineurin-deficient cells. By analysis of a systematic deletion library, we found 48 genes whose mutation resulted in increased sensitivity to the calcineurin inhibitor FK506. Twenty of these mutations (42%) also provoked alkaline pH sensitivity. In conclusion, our results demonstrated that calcium signaling and calcineurin activation represented a significant component of the yeast response to environmental alkalinization.Calcium-mediated signaling mechanisms are used by virtually every eukaryotic cell to regulate a wide variety or cellular processes, including gene expression. Transient increases in cytosolic calcium results in activation of diverse enzymes, such as the protein phosphatase calcineurin. Calcineurin is a heterodimer of catalytic subunit and regulatory subunits. In the yeast Saccharomyces cerevisiae, the catalytic subunit is encoded by two genes, CNA1 and CNA2 (1), whereas a single gene, CNB1, encodes the regulatory subunit (2). Cells lacking the catalytic subunits, or the regulatory subunit, are deficient in calcineurin activity.Exposure of yeast cells to a number of signals, such as ␣-factor (3, 4), glucose (5), sphingosine (6), and certain stress conditions (7-9), triggers a rise in cytoplasmic calcium. This increase in calcium can be a consequence of external calcium influx or release from internal stores, such as the vacuole, and results in activation of calcineurin. For instance, hyperosmotic shock has been reported to provoke calcium release from vacuolar stores (8) through Yvc1, a member of the transient receptor potential channel family, and to trig...

Section: Table V Evaluation By Dna Microarray and Quantitative Real Tsupporting

confidence: 63%

Section: Table V Evaluation By Dna Microarray and Quantitative Real Tmentioning

confidence: 59%

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Characterization of the Calcium-mediated Response to Alkaline Stress in Saccharomyces cerevisiae

Viladevall¹,

Serrano²,

Ruiz³

et al. 2004

188

212

“…An important conclusion from this work is that SOS2 may have functions in plant signaling that are independent of SOS3 and SOS1 because we demonstrate that SOS2 may also act in an SOS3-independent manner to directly regulate the vacuolar H ϩ /Ca 2ϩ antiporter CAX1. In yeast, hyperosmotic stress caused by NaCl and other stresses induce an immediate increase in cytosolic Ca 2ϩ levels that activate the phosphatase calcineurin to mediate ion homeostasis and salt tolerance (26,27). Calcineurin is composed of a regulatory and catalytic subunit that is stimulated by Ca 2ϩ / calmodulin (28) to regulate many transporters including the vacuolar H ϩ /Ca 2ϩ transporter VCX1 (10).…”

Section: Discussionmentioning

confidence: 99%

The Protein Kinase SOS2 Activates the Arabidopsis H+/Ca2+ Antiporter CAX1 to Integrate Calcium Transport and Salt Tolerance

Cheng

Pittman

Zhu

et al. 2004

229

149

“…These non-pore-forming subunits dramatically influence the properties and surface expression of the channels (12)(13)(14)(15)(16). Although the channel activity of Cch1 has not yet been revealed experimentally and Mid1 has no homology to the auxiliary subunits, Mid1 has been shown to cooperate with Cch1 in mating pheromone-induced Ca 2ϩ uptake (8,9,17), store-operated or capacitative Ca 2ϩ entry (10), endoplasmic reticulum stress-induced Ca 2ϩ uptake (18), and a hyperosmotic stress-induced increase in cytosolic Ca 2ϩ (19). On the other hand, it has been shown recently that Mid1, but not Cch1, is required for an antiarrhythmic drug amiodarone-induced increase in cytosolic Ca 2ϩ mainly caused by Ca 2ϩ influx (20).…”

mentioning

confidence: 99%

Molecular Dissection of the Hydrophobic Segments H3 and H4 of the Yeast Ca2+ Channel Component Mid1

Tada¹,

Ohmori²,

Iida³

2003

The Saccharomyces cerevisiae MID1 gene product, Mid1, is composed of 548 amino acid residues, has four relatively hydrophobic segments named H1-H4, and functions as a Ca 2؉ -permeable, stretch-activated channel when expressed in mammalian cells. In some conditions Mid1 cooperates with Cch1, a yeast homolog of the ␣1 subunit of mammalian voltage-gated channels. To identify the important regions or amino acid residues necessary for Mid1 function, we employed in vitro sitedirected mutagenesis on H3 and H4 of Mid1 and expressed the resulting mutant genes in a mid1 null mutant to examine whether the mutant gene products are functional or not in vivo. Mutant Mid1 proteins lacking the whole H3 or H4 segment, H3De or H4De, did not complement the lethality and low Ca 2؉ accumulation activity of the mid1 mutant, although their localization and contents appeared to be normal, indicating that H3 and H4 are required for Mid1 function itself. Single amino acid exchange experiments on individual amino acid residues of H3 and H4 showed that 10 of 20 residues in H3 and 14 of 23 residues in H4 were important for the normal function of Mid1. In particular, we found four severe loss-of-function mutations, D341E, F356S, C373D, and C373R, and two interesting mutations leading to a high level of Ca 2؉ accumulation with a slightly low complementing activity, G342A and Y355A. The importance of these amino acid residues will be discussed.