Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)-Universidad de Buenos Aires, Instituto de Química y Fisicoquímica Biológicas (IQUIFIB), Universidad de Buenos Aires, Buenos Aires, Argentina.
Department of Plant and Environmental Sciences, University of Copenhagen, Copenhagen, Denmark.
PLoS One. 2020 Apr 30;15(4):e0232476. doi: 10.1371/journal.pone.0232476. eCollection 2020.
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.
P5 ATPases 是真核生物中对细胞内金属离子、脂质和蛋白质稳态至关重要的泵；然而，其转运的底物（如果有的话）仍有待确定。因为 Ca2+ 降低了酿酒酵母 Spf1p P5A ATPase 的磷酸化酶水平，所以有人提出 Ca2+ 可以作为 P5 ATPases 的配体。重复以前的纯化方案，我们获得了接近均一的纯化 Spf1p 制剂，并表现出由 CaCl2 刺激的 ATP 水解活性。引人注目的是，催化失活突变体 Spf1p (D487N) 的制剂也表现出 Ca2+ 依赖性 ATP 水解活性。这些结果表明，Spf1p 制剂中含有一种共纯化的蛋白，能够以高速度水解 ATP。该活性可能归因于一种磷酸酶，因为该蛋白：i）当使用 pNPP 作为底物时具有高度活性，ii）需要 Ca2+ 或 Zn2+ 才能发挥活性，iii）强烈被钼酸盐、铍和其他磷酸酶底物抑制。质谱鉴定出磷酸酶 Pho8p 是 Spf1p 制剂的一种污染物。对纯化程序的修改导致了一种无污染物的 Spf1p 制剂，该制剂既不受 Ca2+ 刺激，也不受 EGTA 或钼酸盐抑制。无污染物的 Spf1p 制剂的磷酸化酶水平不受 Ca2+ 的影响。这些结果表明，报道的 Ca2+ 对 Spf1p 的影响不反映 Spf1p 的固有特性，而是由伴随的磷酸酶的活性介导的。
