Molecular heterogeneity of phospholipase D (PLD). Cloning of PLDgamma and regulation of plant PLDgamma, -beta, and -alpha by polyphosphoinositides and calcium.
Phospholipase D (PLD) has emerged as an important enzyme involved in signal transduction, vesicle trafficking, and membrane metabolism. This report describes the cloning and expression of a new Arabidopsis PLD cDNA, designated PLDgamma, and the regulation of PLDgamma, -beta, and -alpha by phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+. The PLDgamma cDNA is 3.3 kilobases in length and codes for an 855-amino acid protein of 95,462 Da with a pI of 6.9. PLDgamma shares a 66% amino acid sequence identity with PLDbeta, but only a 41% identity with PLDalpha. A potential N-terminal myristoylation site is found in PLDgamma, but not in PLDalpha and -beta. Catalytically active PLDgamma was expressed in Escherichia coli, and its activity requires polyphosphoinositides. Both PLDgamma and -beta are most active at microM Ca2+ concentrations, whereas the optimal PLDalpha activity requires mM Ca2+ concentrations. Binding studies showed that the PLDs bound PIP2 in the order of PLDbeta > PLDgamma > PLDalpha. This binding ability correlates with the degree of conservation of a basic PIP2-binding motif located near the putative catalytic site. The binding of [3H]PIP2 was saturable and could be competitively decreased by addition of unlabeled PIP2. Neomycin inhibited the activities of PLDgamma and -beta, but not PLDalpha. These results demonstrate that PLD is encoded by a heterogeneous gene family and that direct polyphosphoinositide binding is required for the activities of PLDgamma and -beta, but not PLDalpha. The different structural and biochemical properties suggest that PLDalpha, -beta, and -gamma are regulated differently and may mediate unique cellular functions.
Qin
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Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506, USA.
Pappan
K
K
Wang
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D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D015139:Blotting, Southern; D002118:Calcium / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D004926:Escherichia coli; D015723:Gene Library; D008969:Molecular Sequence Data; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D010739:Phospholipase D / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D018076:DNA, Complementary; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D010739:Phospholipase D; D002118:Calcium