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© The Rockefeller University Press, 0021-9525/1997//495 $5.00
The Journal of Cell Biology, Volume 138, Number 3, , 1997 495-504

Article

Phospholipase D Stimulates Release of Nascent Secretory Vesicles from the trans-Golgi Network



Ye-Guang Chen*, Anirban Siddhanta*, Cary D. Austin*, Scott M. Hammond§, Tsung-Chang Sung§, Michael A. Frohman§, Andrew J. Morris§, and Dennis Shields*,{ddagger}

* Department of Developmental and Molecular Biology, {ddagger} Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461; and § Department of Pharmacology, and Institute for Cell and Molecular Biology, State University of New York, Stony Brook, New York 11794

Phospholipase D (PLD) is a phospholipid hydrolyzing enzyme whose activation has been implicated in mediating signal transduction pathways, cell growth, and membrane trafficking in mammalian cells. Several laboratories have demonstrated that small GTP-binding proteins including ADP-ribosylation factor (ARF) can stimulate PLD activity in vitro and an ARF-activated PLD activity has been found in Golgi membranes. Since ARF-1 has also been shown to enhance release of nascent secretory vesicles from the TGN of endocrine cells, we hypothesized that this reaction occurred via PLD activation. Using a permeabilized cell system derived from growth hormone and prolactin-secreting pituitary GH3 cells, we demonstrate that immunoaffinity-purified human PLD1 stimulated nascent secretory vesicle budding from the TGN approximately twofold. In contrast, a similarly purified but enzymatically inactive mutant form of PLD1, designated Lys898Arg, had no effect on vesicle budding when added to the permeabilized cells. The release of nascent secretory vesicles from the TGN was sensitive to 1% 1-butanol, a concentration that inhibited PLD-catalyzed formation of phosphatidic acid. Furthermore, ARF-1 stimulated endogenous PLD activity in Golgi membranes approximately threefold and this activation correlated with its enhancement of vesicle budding. Our results suggest that ARF regulation of PLD activity plays an important role in the release of nascent secretory vesicles from the TGN.

Abbreviations used in this paper: ARF, ADP ribosylation factor; BFA, brefeldin A; ERS, energy regenerating system; GAP, GDPase activating protein; GEF, guanosine nucleotide exchange factors; GH, growth hormone; PA, phosphatidic acid; PI-TP, phosphoinositol transfer protein; PKC, protein kinase C; PLD, phospholipase D; PRL, prolactin; PtdBut, phosphatidylbutanol; PtdIns-4,5-P2, phosphatidyl 4,5-bisphosphate.

Please address all correspondence to Dennis Shields, Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Tel.: (718) 430-3306. Fax: (718) 430-8567.

D. Shields thanks A. Elgort for expert technical help and Drs. D. Wilson, M. Kielian, and P. Melançon for many helpful suggestions and discussions.


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