Substitution of the N-terminal segment of the plasma membrane Ca pump isoform 4 by that of isoform 1 results in a fully functional chimeric enzyme

AbstractThe N-terminal segment of the plasma membrane Ca2+ pump (PMCA) is one of the most variable regions among the four isoforms of the enzyme and its functional importance is unknown. In the present work, the N-terminal segment of the highly active C-terminally truncated h4 mutant, h4(ct120) was modified either by substituting residues 18–43 by residues 43–75 or by replacing residues 1–75 by the homologous region from isoform h1 (residues 1–79). Immunoblot analysis of microsomal membranes from transfected COS-1 cells showed that the two N-terminally mutated proteins were correctly expressed at a level similar to that of h4(ct120). Measurements of the Ca2+ uptake by microsomal vesicles from transfected COS-1 cells indicated that mutant (18–43→43–75)h4(ct120) had only negligible Ca2+ transport activity while the chimeric (n1–79)h1h4(ct120) enzyme was fully capable of functioning as a calcium pump.Like h4(ct120), the chimeric mutant was not stimulated further by calmodulin, and was inhibited to a similar degree by the C28R2 peptide corresponding to the calmodulin binding autoinhibitory region of the pump. Moreover, the apparent affinity for Ca2+ and the ATP dependence of the chimeric enzyme were similar to those of the h4(ct120) pump suggesting that the variability of sequence between the N-terminal segment of PMCA isoforms h1 and h4 involves amino acid substitutions that do not substantially change the behavior of the h4 enzyme.Altogether, these results demonstrate that for activity the h4 Ca pump requires a specific amino acid sequence at its N-terminus, and the essential elements for a fully active enzyme can be provided by the N-terminal segment of isoform h1 despite the variability

The Parkinson-associated human P5B-ATPase ATP13A2 protects against the iron-induced cytotoxicity

AbstractP-type ion pumps are membrane transporters that have been classified into five subfamilies termed P1–P5. The ion transported by the P5-ATPases is not known. Five genes named ATP13A1–ATP13A5 that belong to the P5-ATPase group are present in humans. Loss-of-function mutations in the ATP13A2 gene (PARK9, OMIM 610513) underlay a form of Parkinson's disease (PD) known as the Kufor–Rakeb syndrome (KRS), which belongs to the group of syndromes of neurodegeneration with brain iron accumulation (NBIA).Here we report that the cytotoxicity induced by iron exposure was two-fold reduced in CHO cells stably expressing the ATP13A2 recombinant protein (ATP13A2). Moreover, the iron content in ATP13A2 cells was lower than control cells stably expressing an inactive mutant of ATP13A2. ATP13A2 expression caused an enlargement of lysosomes and late endosomes. ATP13A2 cells exhibited a reduced iron-induced lysosome membrane permeabilization (LMP). These results suggest that ATP13A2 overexpression improves the lysosome membrane integrity and protects against the iron-induced cell damage

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

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