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.

Plasma Membrane Ca2+ Pump PMCA4z Is More Active Than Splicing Variant PMCA4x.

The plasma membrane Ca2+ pumps (PMCA) are P-ATPases that control Ca2+ signaling and homeostasis by transporting Ca2+ out of the eukaryotic cell. Humans have four genes that code for PMCA isoforms (PMCA1-4). A large diversity of PMCA isoforms is generated by alternative mRNA splicing at sites A and C. The different PMCA isoforms are expressed in a cell-type and developmental-specific manner and exhibit differential sensitivity to a great number of regulatory mechanisms. PMCA4 has two A splice variants, the forms "x" and "z". While PMCA4x is ubiquitously expressed and relatively well-studied, PMCA4z is less characterized and its expression is restricted to some tissues such as the brain and heart muscle. PMCA4z lacks a stretch of 12 amino acids in the so-called A-M3 linker, a conformation-sensitive region of the molecule connecting the actuator domain (A) with the third transmembrane segment (M3). We expressed in yeast PMCA4 variants "x" and "z", maintaining constant the most frequent splice variant "b" at the C-terminal end, and obtained purified preparations of both proteins. In the basal autoinhibited state, PMCA4zb showed a higher ATPase activity and a higher apparent Ca2+ affinity than PMCA4xb. Both isoforms were stimulated by calmodulin but PMCA4zb was more strongly activated by acidic lipids than PMCA4xb. The results indicate that a PMCA4 intrinsically more active and more responsive to acidic lipids is produced by the variant "z" of the splicing site A.

The Spf1p P5A-ATPase "arm-like" domain is not essential for ATP hydrolysis but its deletion impairs autophosphorylation.

The yeast Spf1p P5A-ATPase actively translocates membrane spanning peptides of mislocalized proteins from the endoplasmic reticulum. Loss of Spf1p function causes a pleiotropic ER stress-phenotype associated with alterations of homeostasis of metal ions, lipids, protein folding, glycosylation, and membrane insertion. A unique characteristic of P5A-ATPases is the presence of an extended insertion which was called the "arm-like" domain connecting the phosphorylation domain (P) with transmembrane segment M5 near the peptidyl-substrate binding pocket. Here we have constructed and characterized a Δarm mutant of Spf1p lacking a segment of 117 amino acids of the "arm-like" domain. The Δarm mutant was capable of hydrolyzing ATP at maximal rates of 50% of that of the wild type enzyme. With the non-nucleotide substrate analog pNPP, the hydrolytic activity of the mutant dropped to 10%. The mutant showed an apparent affinity for ATP similar to the wild type. When incubated with ATP the Δarm mutant produced a lower level of the catalytic phosphoenzyme in amounts proportionate to the ATPase activity. These results indicate that the "arm-like" domain is not essential for hydrolytic activity and suggest that it is needed for the stabilization of Spf1p in a phosphorylation-ready conformation.

Highly exposed segment of the Spf1p P5A-ATPase near transmembrane M5 detected by limited proteolysis.

The yeast Spf1p protein is a primary transporter that belongs to group 5 of the large family of P-ATPases. Loss of Spf1p function produces ER stress with alterations of metal ion and sterol homeostasis and protein folding, glycosylation and membrane insertion. The amino acid sequence of Spf1p shows the characteristic P-ATPase domains A, N, and P and the transmembrane segments M1-M10. In addition, Spf1p exhibits unique structures at its N-terminus (N-T region), including two putative additional transmembrane domains, and a large insertion connecting the P domain with transmembrane segment M5 (D region). Here we used limited proteolysis to examine the structure of Spf1p. A short exposure of Spf1p to trypsin or proteinase K resulted in the cleavage at the N and C terminal regions of the protein and abrogated the formation of the catalytic phosphoenzyme and the ATPase activity. In contrast, limited proteolysis of Spf1p with chymotrypsin generated a large N-terminal fragment containing most of the M4-M5 cytosolic loop, and a minor fragment containing the C-terminal region. If lipids were present during chymotryptic proteolysis, phosphoenzyme formation and ATPase activity were preserved. ATP slowed Spf1p proteolysis without detectable changes of the generated fragments. The analysis of the proteolytic peptides by mass spectrometry and Edman degradation indicated that the preferential chymotryptic site was localized near the cytosolic end of M5. The susceptibility to proteolysis suggests an unexpected exposure of this region of Spf1p that may be an intrinsic feature of P5A-ATPases.

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.