Ca2+ induces spontaneous dephosphorylation of a novel P5A-type ATPase.
P5 ATPases constitute the least studied group of P-type ATPases, an essential family of ion pumps in all kingdoms of life. Although P5 ATPases are present in every eukaryotic genome analyzed so far, they have remained orphan pumps, and their biochemical function is obscure. We show that a P5A ATPase from barley, HvP5A1, locates to the endoplasmic reticulum and is able to rescue knock-out mutants of P5A genes in both Arabidopsis thaliana and Saccharomyces cerevisiae. HvP5A1 spontaneously forms a phosphorylated reaction cycle intermediate at the catalytic residue Asp-488, whereas, among all plant nutrients tested, only Ca(2+) triggers dephosphorylation. Remarkably, Ca(2+)-induced dephosphorylation occurs at high apparent [Ca(2+)] (K(i) = 0.25 mM) and is independent of the phosphatase motif of the pump and the putative binding site for transported ligands located in M4. Taken together, our results rule out that Ca(2+) is a transported substrate but indicate the presence of a cytosolic low affinity Ca(2+)-binding site, which is conserved among P-type pumps and could be involved in pump regulation. Our work constitutes the first characterization of a P5 ATPase phosphoenzyme and points to Ca(2+) as a modifier of its function.
Centre for Membrane Pumps in Cells and Disease, PUMPKIN, Danish National Research Foundation, Department of Plant Biology and Biotechnology, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.
Danny Mollerup
DM
Sørensen
Annette B
AB
Møller
Mia K
MK
Jakobsen
Michael K
MK
Jensen
Peter
P
Vangheluwe
Morten J
MJ
Buch-Pedersen
Michael G
MG
Palmgren
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D055786:Gene Knockout Techniques; D005816:Genetic Complementation Test; D001467:Hordeum / Q000201:enzymology* / Q000235:genetics; D010766:Phosphorylation / Q000502:physiology; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics
D010940:Plant Proteins; D000251:Adenosine Triphosphatases; D002118:Calcium