Mutant <i>cta4Δ</i> displays higher calcineurin phosphatase activity.

<p>The calcineurin protein phosphatase activity was determined by the amount of free phosphate released. The reaction was started by adding the cellular extracts, followed by incubation at 30°C for 30 min with the RII phosphopeptide substrate. Values are means (± SE) of three independent experiments.</p

⁴⁵Ca²⁺ uptake by total membranes of <i>S. pombe</i> is mediated by Ca²⁺- ATPases and Ca²⁺/H⁺ exchangers.

<p>Total membranes were isolated from wild type (▪,□) and <i>cta4Δ</i> (•,○) cells and subjected to determination of ⁴⁵Ca²⁺ transport in the presence of 1 mM ATP and in the presence (□,○) or absence (▪,•) of 2 µM FCCP (A). Inhibition of ATP-dependent FCCP-insensitive ⁴⁵Ca²⁺ transport by vanadate (Na₃VO₄), the inhibitor of P-type ATPases (B). Values are means (± SE) of four independent experiments.</p

Loss of <i>cta4⁺</i> results in elevated cellular Ca²⁺ levels.

<p>⁴⁵Ca²⁺ accumulation in wild-type cells and mutant cells lacking Cta4 ATPase was measured after incubation for 5 hr in standard medium supplemented with ⁴⁵Ca²⁺ as described in Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027843#s2" target="_blank">Methods</a>. Values are means (± SE) of three independent experiments.</p

Cells lacking Cta4p exhibited higher levels of the ER stress indicator, BiP.

<p>Total membranes (TM) were isolated from yeast cells, fractionated on a sucrose density gradient and submitted to immunoblots analysis as described in Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027843#s2" target="_blank">Methods</a>. Dot blots of individual fractions (10 µL, numbers are indicated) were used. Abbreviation used: NE, nuclear envelope; ER, endoplasmic reticulum; Vac, vacuole.</p

Endoplasmic reticulum stress caused by inhibition of glycosylation stimulates an increase of Ca²⁺ accumulation.

<p>The accumulation of ⁴⁵Ca²⁺ in wild-type and <i>cta4Δ</i> yeast cells was measured after 5 hours of addition of ⁴⁵Ca²⁺ to YES medium containing 0.125 µg/mL tunicamycin. Values are means (± SE) of three independent experiments.</p

Calcineurin inhibition stimulated ⁴⁵Ca²⁺ accumulation in wt and <i>cta4Δ</i> mutant cells.

<p>The accumulation of ⁴⁵Ca²⁺ in wild-type and <i>cta4Δ</i> yeast cells was measured after 5 hours of addition of ⁴⁵Ca²⁺ to YES medium containing 10 µg/mL cyclosporin A (CsA). Values are means (± SE) of three independent experiments.</p

The growth of <i>cta4Δ</i> is impaired by endoplasmic reticulum stress.

<p>Wild-type and <i>cta4Δ</i> cells were serially diluted in five-fold steps, spotted onto YES plates containing 0.4% DTT and 0.05 µg/mL tunicamycin and incubated for 3 days at 30°C.</p

<i>cta4⁺</i> is required for Ca²⁺-ATPase activity in ER membrane fractions.

<p>Total membranes were isolated from wt and <i>cta4Δ</i> cells and fractionated on 12-step sucrose density gradient. (A) ATP-dependent FCCP-insensitive ⁴⁵Ca²⁺ uptake in the membrane fractions was measured after 10 min of incubation as described in Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027843#s2" target="_blank">Methods</a>. Sucrose concentration of each fraction is shown. (B) Dotblot of selected gradient fractions (fraction numbers are indicated) was used for immunolocalization of Cta4-GFP using anti-GFP antibodies. (C) Inhibition of ATP-dependent FCCP-insensitive ⁴⁵Ca²⁺ transport in ER membrane fractions by vanadate (Na₃VO₄), the inhibitor of P-type ATPases. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027843#s3" target="_blank">Results</a> shown are representative of three independent experiments. Abbreviation used: NE, nuclear envelope; ER, endoplasmic reticulum; Vac, vacuole.</p

Comparison of properties of the V H⁺-ATPase activated and non-activated by extracellular glucose.

<p>(<b>a</b>) Extracellular glucose causes asymmetrical increase in the immune reactivity of subunits A and B as well as in the initial velocities of H⁺ transport and ATP hydrolysis. The protein content of these subunits was not changed in contradiction with the mechanistic model prediction (Fig. 1, step A). (<b>b</b>) Protein profiles of TM isolated from the spheroplasts pre-incubated with and without 100 mM extracellular glucose. The proteins were solubilised with 10% SDS, separated by SDS-PAGE (15 µg/each well; see Methods) and stained with Coomassie blue. The representative data from five independent isolations of TM are shown. The Arabic numbers indicate the stimulation degree of the initial velocities of H⁺ transport for each experiment. (<b>c</b>) Immunoblot of activated and non-activated enzyme; the amount of 30 µg and 15 µg of protein were loaded onto the wells corresponding to (−) and (+) glucose, respectively. (<b>d</b>) After SDS-PAGE, the protein bands were firstly visualised with Coomassie blue and, after gel destaining, were secondly detected by silver stain. Protein content loaded onto the wells 3−;3+;4− and 4+( = 5−;5+ and 6−;6+) was 7, 11, 13 and 15 µg, respectively. A merger of the Coomasie-stained gel and immunoblot was used to localise the bands corresponding to the subunits A and B in each experiment (not shown). One representative experiment of five independent membrane isolations is shown.</p

A model for the hypothetical regulation of V H⁺-ATPase activity by extracellular glucose.

<p>Step A: the dissociation and reassociation of the catalytic complex V₁, according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049580#pone.0049580-Sumner1" target="_blank">[17]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049580#pone.0049580-Kane1" target="_blank">[18]</a>; alternative step B: a putative biochemical modification and conformational change of V₁ subunits (this report).</p