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Published online 16 December 2002. doi:10.1083/jcb.200205056
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© The Rockefeller University Press, 0021-9525/2002/12/1039 $5.00
The Journal of Cell Biology, Volume 159, Number 6, 1039-1049

Article

Dual role for phosphoinositides in regulation of yeast and mammalian phospholipase D enzymes



Vicki A. Sciorra1, Simon A. Rudge1, Jiyao Wang2, Stuart McLaughlin2, JoAnne Engebrecht3 and Andrew J. Morris4

1 Howard Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093
2 Department of Physiology and Biophysics, State University of New York at Stony Brook, Stony Brook, NY 11794
3 Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY 11794
4 Department of Cell and Developmental Biology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599

Address correspondence to Andrew J. Morris, Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, 522 Taylor Hall, CB# 7090, Chapel Hill, NC 27599-7090. Tel.: (919) 843-5001. Fax: (919) 966-1856. E-mail: ajmorris{at}med.unc.edu

Phospholipase D (PLD) generates lipid signals that coordinate membrane trafficking with cellular signaling. PLD activity in vitro and in vivo is dependent on phosphoinositides with a vicinal 4,5-phosphate pair. Yeast and mammalian PLDs contain an NH2-terminal pleckstrin homology (PH) domain that has been speculated to specify both subcellular localization and regulation of PLD activity through interaction with phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2). We report that mutation of the PH domains of yeast and mammalian PLD enzymes generates catalytically active PI(4,5)P2-regulated enzymes with impaired biological functions. Disruption of the PH domain of mammalian PLD2 results in relocalization of the protein from the PI(4,5)P2-containing plasma membrane to endosomes. As a result of this mislocalization, mutations within the PH domain render the protein unresponsive to activation in vivo. Furthermore, the integrity of the PH domain is vital for yeast PLD function in both meiosis and secretion. Binding of PLD2 to model membranes is enhanced by acidic phospholipids. Studies with PLD2-derived peptides suggest that this binding involves a previously identified polybasic motif that mediates activation of the enzyme by PI(4,5)P2. By comparison, the PLD2 PH domain binds PI(4,5)P2 with lower affinity but sufficient selectivity to function in concert with the polybasic motif to target the protein to PI(4,5)P2-rich membranes. Phosphoinositides therefore have a dual role in PLD regulation: membrane targeting mediated by the PH domain and stimulation of catalysis mediated by the polybasic motif.

Key Words: phospholipases; signal transduction; phosphatidylinositol phosphates; GTP-binding proteins; membrane lipids


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