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 K_1/2 = 14 and 800 μM Ca²⁺, Mn²⁺ and Co²⁺ lowered about 50% of the Spf1p ATPase with relatively low affinity (K_i ∼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.

• PMID: 36328152
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Journal Article | Research Support, Non-U.S. Gov't

Authors

Petrovich GD, Corradi GR, Adamo HP

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