Dual mechanism of activation of plant plasma membrane Ca2+-ATPase by acidic phospholipids: evidence for a phospholipid binding site which overlaps the calmodulin-binding site.
The effect of phospholipids on the activity of isoform ACA8 of Arabidopsis thaliana plasma membrane (PM) Ca2+-ATPase was evaluated in membranes isolated from Saccharomyces cerevisiae strain K616 expressing wild type or mutated ACA8 cDNA. Acidic phospholipids stimulated the basal Ca2+-ATPase activity in the following order of efficiency: phosphatidylinositol 4-monophosphate > phosphatidylserine > phosphatidylcholine approximately = phosphatidylethanolamine approximately = 0. Acidic phospholipids increased V(max-Ca2+) and lowered the value of K(0.5-Ca2+) below the value measured in the presence of calmodulin (CaM). In the presence of CaM acidic phospholipids activated ACA8 by further decreasing its K(0.5-Ca2+) value. Phosphatidylinositol 4-monophosphate and, with lower efficiency, phosphatidylserine bound peptides reproducing ACA8 N-terminus (aa 1-116). Single point mutation of three residues (A56, R59 and Y62) within the sequence A56-T63 lowered the apparent affinity of ACA8 for phosphatidylinositol 4-monophosphate by two to three fold, indicating that this region contains a binding site for acidic phospholipids. However, the N-deleted mutant Delta74-ACA8 was also activated by acidic phospholipids, indicating that acidic phospholipids activate ACA8 through a complex mechanism, involving interaction with different sites. The striking similarity between the response to acidic phospholipids of ACA8 and animal plasma membrane Ca2+-ATPase provides new evidence that type 2B Ca2+-ATPases share common regulatory properties independently of structural differences such as the localization of the terminal regulatory region at the N- or C-terminal end of the protein.
Meneghelli
Silvia
S
Dipartimento di Biologia, Universita di Milano, Istituto di Biofisica del CNR, Sezione di Milano, Milano, Italy.
Fusca
Tiziana
T
Luoni
Laura
L
De Michelis
Maria Ida
MI
D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D004789:Enzyme Activation; D008565:Membrane Proteins; D009154:Mutation; D010743:Phospholipids / Q000378:metabolism* / Q000494:pharmacology; D020033:Protein Isoforms; D012441:Saccharomyces cerevisiae / Q000235:genetics
D029681:Arabidopsis Proteins; D002147:Calmodulin; D008565:Membrane Proteins; D010743:Phospholipids; D020033:Protein Isoforms; C508150:Aca8 protein, Arabidopsis; D000252:Calcium-Transporting ATPases