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Published 21 July 2003. doi:10.1083/jcb.200302033
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© The Rockefeller University Press, 0021-9525/2003/7/305 $5.00
The Journal of Cell Biology, Volume 162, Number 2, 305-315

Article

Regulation of phospholipase D1 subcellular cycling through coordination of multiple membrane association motifs



Guangwei Du1, Yelena M. Altshuller1, Nicolas Vitale2, Ping Huang1, Sylvette Chasserot-Golaz2, Andrew J. Morris1, Marie-France Bader2 and Michael A. Frohman1

1 Department of Pharmacology and the Center for Developmental Genetics, University Medical Center, State University of New York at Stony Brook, Stony Brook, NY 11794
2 Centre National de la Recherche Scientifique, Unité Propre de Recherche 2356, IFR 37, 67084 Strasbourg Cedex, France

Address correspondence to Michael A. Frohman, Dept. of Pharmacology and the Center for Developmental Genetics, 438 CMM, State University of New York at Stony Brook, Stony Brook, NY 11794-5140. Tel.: (631) 632-1476. Fax: (631) 632-1692. E-mail: michael{at}pharm.sunysb.edu

The signaling enzyme phospholipase D1 (PLD1) facilitates membrane vesicle trafficking. Here, we explore how PLD1 subcellular localization is regulated via Phox homology (PX) and pleckstrin homology (PH) domains and a PI4,5P2-binding site critical for its activation. PLD1 localized to perinuclear endosomes and Golgi in COS-7 cells, but on cellular stimulation, translocated to the plasma membrane in an activity-facilitated manner and then returned to the endosomes. The PI4,5P2-interacting site sufficed to mediate outward translocation and association with the plasma membrane. However, in the absence of PX and PH domains, PLD1 was unable to return efficiently to the endosomes. The PX and PH domains appear to facilitate internalization at different steps. The PH domain drives PLD1 entry into lipid rafts, which we show to be a step critical for internalization. In contrast, the PX domain appears to mediate binding to PI5P, a lipid newly recognized to accumulate in endocytosing vesicles. Finally, we show that the PH domain–dependent translocation step, but not the PX domain, is required for PLD1 to function in regulated exocytosis in PC12 cells. We propose that PLD1 localization and function involves regulated and continual cycling through a succession of subcellular sites, mediated by successive combinations of membrane association interactions.

Key Words: membrane localization; Phox homology domain; pleckstrin homology domain; membrane trafficking; phospholipase D

A.J. Morris' present address is Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, NC 27599-7090.

* Abbreviations used in this paper: hGH, human growth hormone; PA, phosphatidic acid; PH, pleckstrin homology; PLD, phospholipase D; PM, plasma membrane; PX, Phox homology; TfR, transferrin receptor.


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