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. 2020 Apr 30;15(4):e0232476.
doi: 10.1371/journal.pone.0232476. eCollection 2020.

Reduction of the P5A-ATPase Spf1p phosphoenzyme by a Ca2+-dependent phosphatase

Gerardo R Corradi  1 , Luciana R Mazzitelli  1 , Guido D Petrovich  1 , Paula Grenon  1 , Danny M Sørensen  2 , Michael Palmgren  2 , Felicitas de Tezanos Pinto  1 , Hugo P Adamo  1
Affiliations

Reduction of the P5A-ATPase Spf1p phosphoenzyme by a Ca2+-dependent phosphatase

Gerardo R Corradi et al. PLoS One. .

Abstract

P5 ATPases are eukaryotic pumps important for cellular metal ion, lipid and protein homeostasis; however, their transported substrate, if any, remains to be identified. Ca2+ was proposed to act as a ligand of P5 ATPases because it decreases the level of phosphoenzyme of the Spf1p P5A ATPase from Saccharomyces cerevisiae. Repeating previous purification protocols, we obtained a purified preparation of Spf1p that was close to homogeneity and exhibited ATP hydrolytic activity that was stimulated by the addition of CaCl2. Strikingly, a preparation of a catalytically dead mutant Spf1p (D487N) also exhibited Ca2+-dependent ATP hydrolytic activity. These results indicated that the Spf1p preparation contained a co-purifying protein capable of hydrolyzing ATP at a high rate. The activity was likely due to a phosphatase, since the protein i) was highly active when pNPP was used as substrate, ii) required Ca2+ or Zn2+ for activity, and iii) was strongly inhibited by molybdate, beryllium and other phosphatase substrates. Mass spectrometry identified the phosphatase Pho8p as a contaminant of the Spf1p preparation. Modification of the purification procedure led to a contaminant-free Spf1p preparation that was neither stimulated by Ca2+ nor inhibited by EGTA or molybdate. The phosphoenzyme levels of a contaminant-free Spf1p preparation were not affected by Ca2+. These results indicate that the reported effects of Ca2+ on Spf1p do not reflect the intrinsic properties of Spf1p but are mediated by the activity of the accompanying phosphatase.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. SDS-PAGE of purified Spf1p and Spf1p (D487N) preparations.
Different volumes of the final 150 mM imidazole eluates of Ni-NTA-purified preparations of Spf1p and Spf1p (D487N) were loaded in each well of a 9% SDS-PAGE gel and stained with Coomassie Blue. The estimated μg of Spf1p protein loaded is indicated.
Fig 2
Fig 2. ATP hydrolysis by Spf1p and Spf1p (D487N) preparations obtained using the standard purification protocol.
The ATPase reaction was performed at 28°C as described in “Materials and Methods” in reaction media containing 50 mM Tris–HCl (pH 7.2 at 28°C), 0.5 mM EGTA, 5 mM N3Na, 2 mM Mg2+, 3 mM ATP, and 1 μg of Spf1p in 40 μL of elution buffer supplemented with 40 μg of C12E10 and 40 μg of PC. The reaction time was 20 min. Where indicated, CaCl2 was added to the reaction medium to yield 10 μM free Ca2+. The data points are from five determinations conducted in duplicate from five independent preparations. Error bars show the standard deviation. At the 0.05 level the mean activities in media with EGTA and EGTA-Ca are significantly different.
Fig 3
Fig 3. Contaminant ATPase and pNPPase activities in a standard preparation of the inactive mutant Spf1p(D487N) obtained by the standard purification protocol.
Panel A- ATPase activity. The reaction medium was similar to that described in Fig 2 except that EGTA was not present except when indicated. CaCl2 and ZnCl2 were added to yield 10 μM and 1 μM of the free ions, respectively. Mo, 1 mM ammonium molybdate, Be, 25 μM beryllium sulfate. The ATPase activity of 2.2 μmol/mg/min without additions (-) was taken as 100%. Panel B- Effect of increasing concentrations of ammonium molybdate on the contaminant ATPase activity. Panel C- pNPPase activity. The hydrolysis of pNPP was measured in the absence or presence of 0.5 mM EGTA. The continuous lines represent fitting to an equation that considers biphasic activation and inhibition components. The K0.5 for pNPP of the activation phase was 0.10 ± 0.05 mM for both conditions with and without EGTA. Panel D- Inhibition of the contaminant ATPase by phosphatase substrates. The measurement was done as indicated in “Material and Methods” in the presence of 10 μM Ca2+. The contaminant ATPase activity at 30 μM ATP concentration was taken as 100%. G6P, glucose 6-phosphate 200 μM, FBP, fructose 1,6 biphosphate 200 μM, pNPP, p-nitrophenyl phosphate 500 μM, PP, phenyl phosphate 500 μM, Pi, inorganic phosphate 3 mM. The reaction time was 20 min. Error bars show the standard deviation.
Fig 4
Fig 4. Phosphoenzyme formation by the “contaminated” Spf1p preparation.
Spf1p (1.5 μg) was suspended at 4°C in a medium containing 2 mM Mg2+, and 0.5 mM EGTA or 0.5 mM EGTA plus enough CaCl2 to give a final concentration of 10 μM Ca2+. The reaction was started by adding 0.5 μM ATP. Where indicated, 1 mM ammonium molybdate was added to the reaction media. The reaction time was 15 s. Panel A- Acidic gel electrophoresis of phosphorylated Spf1p showing the radioactivity. Panel B- Coomassie Blue stained gel. Panel C- EP levels quantified as described under “Materials and Methods”. Duplicates for each condition are shown. The EP level detected in the EGTA reaction medium was taken as 100%. Error bars show the standard deviation.
Fig 5
Fig 5. ATPase activity and sensitivity to inhibition by molybdate of the pure Spf1p and Spf1p (D487N).
Panel A- ATP hydrolysis was estimated as indicated in “Materials and Methods”. The composition of the reaction medium was the same as that indicated in Fig 2. The reaction time was 20 min. Error bars show the standard deviation. At the 0.05 level the mean activities in media with EGTA and EGTA-Ca are not significantly different. Panel B- Effect of increasing concentrations of ammonium molybdate on the ATPase activity of pure Spf1p. The activity at 0 mM molybdate was taken as 100%.
Fig 6
Fig 6. Phosphoenzyme formation by the pure Spf1p preparation.
Spf1p (1.5 μg) was phosphorylated under identical conditions as those described in Fig 4. Panel A- Acidic gel electrophoresis. Panel B- Coomassie Blue-stained gel. Panel C- Quantitation of EP. Duplicates for each condition are shown. The EP level detected in the EGTA reaction medium was taken as 100%. Error bars show the standard deviation.
Fig 7
Fig 7. Comparison of Pho8p content of standard and pure Spf1p preparations.
Top, SDS-PAGE of standard and pure Spf1p preparations. The μg of Spf1p protein loaded in each lane is indicated. M, molecular weight marker, y, 20 μg of yeast microsomal protein. Bottom, Western blot using antiPho8p antibody.
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This work was supported by Grant PICT 1690 from Agencia Nacional de Promoción Científica y Tecnológica (HPA), Grant PIP 773 from Consejo Nacional de Investigaciones Científicas y Tecnológicas (HPA), and UBACyT from Universidad de Buenos Aires (HPA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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