P5A-Type ATPase Cta4p Is Essential for Ca2+ Transport in the Endoplasmic Reticulum of Schizosaccharomyces pombe

This study establishes the role of P5A-type Cta4 ATPase in Ca2+ sequestration in the endoplasmic reticulum by detecting an ATP-dependent, vanadate-sensitive and FCCP insensitive 45Ca2+-transport in fission yeast membranes isolated by cellular fractionation. Specifically, the Ca2+-ATPase transport activity was decreased in ER membranes isolated from cells lacking a cta4+ gene. Furthermore, a disruption of cta4+ resulted in 6-fold increase of intracellular Ca2+ levels, sensitivity towards accumulation of misfolded proteins in ER and ER stress, stimulation of the calcineurin phosphatase activity and vacuolar Ca2+ pumping. These data provide compelling biochemical evidence for a P5A-type Cta4 ATPase as an essential component of Ca2+ transport system and signaling network which regulate, in conjunction with calcineurin, the ER functionality in fission yeast

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

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

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