Neuronal calcium sensor-1 (Ncs1p) is up-regulated by calcineurin to promote Ca2+ tolerance in fission yeast

Abstract

Neuronal calcium sensor (NCS) proteins regulate signal transduction and are highly conserved from yeast to humans. NCS homolog in fission yeast (Ncs1p) is essential for cell growth under extreme Ca(2+) conditions. Ncs1p expression increases approximately 100-fold when fission yeast grows in high extracellular Ca(2+) (>0.1 M). Here, we show that Ca(2+)-induced expression of Ncs1p is controlled at the level of transcription. Transcriptional reporter assays show that ncs1 promoter activity increased 30-fold when extracellular Ca(2+) was raised to 0.1 M and was highly Ca(2+)-specific. Ca(2+)-dependent transcription of ncs1 is abolished by the calcineurin inhibitor (FK506) and by knocking out the calcineurin target, prz1. Thus, Ca(2+)-induced expression of Ncs1p is linked to the calcineurin/prz1 stress response. The Ca(2+)-responsive ncs1 promoter region consists of 130 nucleotides directly upstream from the start codon and contains tandem repeats of the sequence, 5'-caact-3', that binds to Prz1p. The Ca(2+)-sensitive ncs1Delta phenotype is rescued by a yam8 null mutation, suggesting a possible interaction between Ncs1p and the Ca(2+) channel, Yam8p. Ca(2+) uptake and Ncs1p binding to yeast membranes are both decreased in yam8Delta, suggesting Ca(2+)-induced binding of Ncs1p to Yam8p results in channel closure. We propose that Ncs1p promotes Ca(2+) tolerance in fission yeast, in part by cytosolic Ca(2+) buffering and perhaps by negatively regulating the Yam8p Ca(2+) channel.

FIGURE 1.

Ncs1 promoter activity regulated by calcium. A, the S. pombe wild-type strain that bears pREP42-mec803-EGFP was grown in EMM-U media containing 0, 0.01, or 0.1 m calcium chloride for 16 h and observed by fluorescence microscopy (×400). The black bar indicates 10 μm. B, the same cells used in A were lysed, and the cell lysates were analyzed by Western blot analysis using anti-GFP antibody (right panel). The left panel shows an equivalent gel stained by Coomassie Brilliant Blue to show that each lane contained the same level of protein.

FIGURE 2.

Calcium specific up-regulation of ncs1 promoter. Reporter activities, using wild-type cells bearing a plasmid pREP-mec803-luxAB (bacterial luciferase) grown in minimum media, were measured 6 h after the cells were treated with various reagents. Promoter activity was shown as a ratio of the luminometer readings of the cells with reagent versus without reagent (see “Experimental Procedures”).

FIGURE 3.

Both ncs1Δ and prz1⁻ mutants regain calcium tolerance in the presence of FK506. Ncs1D and prz1⁻ mutant cells (two independent isolates for prz1⁻) were streaked on rich media agar plates containing 0.1 m calcium chloride and/or 0.5 μg/ml FK506.

FIGURE 4.

Membrane-binding pulldown assay. Plasma membrane fractions from S. pombe wild-type and yam8 deletion mutant (KP2758) cells were treated with recombinant, unmyristoylated Ncs1p in the presence (right) and absence (left) of saturating Ca²⁺. Upper panel, Ncs1p bound to S. pombe membranes probed by Western blot analysis. Bottom panel, SDS-PAGE of membrane fractions visualized by Coomassie Brilliant Blue staining.

FIGURE 5.

Calcium uptake activity in fission yeast. Calcium 45 uptake by yeast cells was measured at different time points after the addition of 1 μm ⁴⁵Ca²⁺ to the cell culture under the same conditions as described in a previous study (64) for wild type (solid squares), ncs1Δ (open triangles), and yam8Δ (cross).

FIGURE 6.

Localization of ncs1 promoter region responsible for calcium-dependent up-regulation. Reporter assay was carried out in the same manner as Fig. 2 except ncs1 promoter mec803 was replaced by mec424, mec206, mec130, mec102, mec78, or mec51. Promoter activation level was indicated by ++ (>20-fold), +/− (5- to 10-fold), and − (<5-fold).

FIGURE 7.

The 130-bp fragment from ncs1 promoter enables high enough expression of ncs1 gene to permit growth of ncs1Δ mutant on agar plate with 0.1 m calcium chloride. The Ncs1Δ mutant strain, which is unable to grow on calcium plates, was transformed with pREP4X-mec424-ncs1, pREP4X-mec130-ncs1, or pREP4X-p102-ncs1, in which nmt1 promoter in pREP-ncs1 was replaced with 424-, 130-, or 102-bp fragments from ncs1 promoter. vector = pREP4X alone; nmt1-ncs1 = original pREP4X (with nmt1 promoter)-ncs1 open reading frame.

FIGURE 8.

Electrophoretic mobility shift assay of Prz1p binding to ncs1 promoter sequence. 5 nm of Cy5 labeled duplex DNA (see “Experimental Procedures”) in 10 mm HEPES (pH 7.5), 0.1 m NaCl, and 5 mm Mg²⁺ was incubated with buffer blank (lane 1) and recombinant Prz1p (lanes 2–5). Cold-chase experiments (see “Experimental Procedures”) were performed using unlabeled wild-type sequence (lane 3), unlabeled random sequence (lane 4), and unlabeled mutant sequence (nucleotides −111 to −107 changed to AGGAC, lane 5).

FIGURE 9.

Schematic model of Ca²⁺-induced expression of ncs1 up-regulated by calcineurin to promote Ca²⁺ tolerance in fission yeast. Extracellular Ca²⁺ enters the cell through Ca²⁺ channels, causing a rise in intracellular Ca²⁺ concentration that activates the calcineurin pathway, leading to Ca²⁺-induced transcription of ncs1. The Ca²⁺-bound form of Ncs1p interacts with the Yam8p Ca²⁺ channel to promote channel closure and thus block the entry of extracellular Ca²⁺ under extreme conditions.