Evidence for and characterization of Ca2+ binding to the catalytic region of Arabidopsis thaliana phospholipase Dbeta.
Most types of plant phospholipase D (PLD) require Ca(2+) for activity, but how Ca(2+) affects PLD activity is not well understood. We reported previously that Ca(2+) binds to the regulatory C2 domain that occurs in the N terminus of the Ca(2+)-requiring PLDs. Using Arabidopsis thaliana PLDbeta and C2-deleted PLDbeta (PLDbetacat), we now show that Ca(2+) also interacts with the catalytic regions of PLD. PLDbetacat exhibited Ca(2+)-dependent activity, was much less active, and required a higher level of Ca(2+) than the full-length PLDbeta. Ca(2+) binding of the proteins was stimulated by phospholipids; phosphatidylserine was the most effective among those tested. Scatchard plot analysis of Ca(2+) binding data yielded an estimate of 3.6 high affinity (K(d) = 29 mum) binding sites on PLDbeta. The Ca(2+)-PLDbetacat interaction increased the affinity of the protein for the activator, phosphatidylinositol 4,5-bisphosphate, but not for the substrate, phosphatidylcholine. This is in contrast to the effect of Ca(2+) binding to the C2 domain, which stimulates phosphatidylcholine binding but inhibits phosphatidylinositol 4,5-bisphosphate binding of the domain. These results demonstrate the contrasting and complementary effects of the Ca(2+)- and lipid-binding properties of the C2 and catalytic domains of plant PLD and provide insight into the mechanism by which Ca(2+) regulates PLD activity.
Pappan
Kirk
K
Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506, USA.
Zheng
Li
L
Krishnamoorthi
Ramaswamy
R
Wang
Xuemin
X
D017360:Arabidopsis / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002132:Calcium Radioisotopes / Q000378:metabolism; D020134:Catalytic Domain; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D010739:Phospholipase D / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D029681:Arabidopsis Proteins; D002132:Calcium Radioisotopes; D007527:Isoenzymes; D010713:Phosphatidylcholines; D019269:Phosphatidylinositol 4,5-Diphosphate; D011993:Recombinant Fusion Proteins; D010739:Phospholipase D; D002118:Calcium