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

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

doi:10.1371/journal.pone.0232476

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.

Highlights

P5 ATPases are the most intriguing members of the large family of P-type ATPase membrane proteins, which couple ATP hydrolysis with the active transport of ligands across biological membranes [1]
On the basis of indirect evidences, previous studies suggested that Ca2+ may be the substrate transported by yeast sensitivity to Pichia farinosa killer toxin protein (Spf1p) P5A-ATPase
A characteristic phenotype of the SPF1 knockout mutant is the disruption of Ca2+ homeostasis and hypersensitivity to external Ca2+, reminiscent of the phenotype of a knockout of the Ca2+ and Mn2+ pump PMR1 [16]

Summary

Introduction

P5 ATPases are the most intriguing members of the large family of P-type ATPase membrane proteins, which couple ATP hydrolysis with the active transport of ligands across biological membranes [1]. In agreement with predictions based on the primary amino acid sequences, all P5A ATPases characterized so far have been shown to hydrolyze ATP and to form an EP phosphoenzyme intermediate [16, 26,27,28]. Ca2 +-dependent EP dephosphorylation did not require the endogenous phosphatase activity of the enzyme [26] and the ATPase activity of Spf1p was only marginally affected by Ca2+ [16, 19, 28]. We found that purified preparations of recombinant His-tagged Spf1p contained trace amounts of a phosphatase that possessed highly active metal ion-dependent ATPase and phosphatase activities. Optimization of the purification procedure caused the Ca2 +-stimulated phosphatase activity to vanish, demonstrating that this activity is not an intrinsic property of the Spf1p enzyme

Materials and methods

Results

Discussion