The effect of metal ions on the Spf1p P5A-ATPase. High sensitivity to irreversible inhibition by zinc

The Spf1p protein from Saccharomyces cerevisiae belongs to the family of P5A-ATPases that have recently been shown to protect the endoplasmic reticulum by dislocating misinserted membrane proteins. The loss of function of P5A-ATPases leads to endoplasmic reticulum stress with a pleiotropic phenotype including protein, sterol and metal ion dyshomeostasis. Like other P-ATPases, Spf1p requires Mg2+. We found that free Mg2+ stimulated the Spf1p ATPase activity along a double hyperbolic curve with two components of K1/2 = 14 and 800 μM Ca2+, Mn2+ and Co2+ lowered about 50% of the Spf1p ATPase with relatively low affinity (Ki ∼75 μM) and the activity was fully recovered after metal ion chelation with EGTA. In contrast, low concentrations of Zn2+ and Cd2+decreased the activity to less than 20% and lead to slow irreversible inactivation of the enzyme. After the treatment with Zn2+, Spf1p exhibited a reduced apparent affinity for ATP and formed lower levels of the catalytic phosphoenzyme. The inactivation by Zn2+ occurred preferentially at a pH > 6 and could be prevented by adding either ATP or ADP to the inactivation media. These results suggest that Zn2+ inactivated Spf1p by binding to amino acid residues from the nucleotide binding-phosphorylation domains that are protonated at lower pH. Alternatively the binding of nucleotides may indirectly compete with a conformational change leading to the Zn2+-inactive form of the enzyme. Exposure of yeast cells to high concentrations of Zn2+ led to changes similar to the phenotype characteristic of the Spf1Δ cells. Altogether, our data, point out a possible mechanism by which the inhibition of P5A-ATPases could potentiate metal ion–induced ER stress and proteotoxicity. [Display omitted] •Spf1p ATPase and Phosphatase activities show a biphasic dependence with Mg2+.•Spf1p ATPase activity is sensitive to divalent metal ions.•Zn2+ and Cd2+ irreversible inhibit Spf1p.•Zn2+-inactivated Spf1p shows lower phosphoenzyme and reduced ATP apparent affinity.•Yeast growing in a medium supplemented with 1 mM ZnCl2 resemble Spf1Δ phenotype.