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

Du G, Altshuller YM, Vitale N, Huang P, Chasserot-Golaz S, Morris AJ, Bader MF, Frohman MA - J. Cell Biol. (2003)

Bottom Line: The PH domain drives PLD1 entry into lipid rafts, which we show to be a step critical for internalization.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University Medical Center, State University of New York at Stony Brook, Stony Brook, NY 11794, USA.

ABSTRACT
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.

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The isolated PLD1 PH domain associates only weakly with internal and plasma membranes, both in resting cells and stimulated cells. COS-7 cells were transfected with plasmids expressing the PH domain flanked by leader and trailing sequences.
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fig7: The isolated PLD1 PH domain associates only weakly with internal and plasma membranes, both in resting cells and stimulated cells. COS-7 cells were transfected with plasmids expressing the PH domain flanked by leader and trailing sequences.

Mentions: The isolated PLD1 PH domain associates with membranes weakly (Fig. 7). It localizes in resting cells to perinuclear membrane structures. With PMA, a very weak translocation is observed to the PM (Fig. 7), suggesting that the PH domain may contribute to a small degree to regulated translocation, although this is minor at best in the context of the full-length protein (as shown in Fig. 3).


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

Du G, Altshuller YM, Vitale N, Huang P, Chasserot-Golaz S, Morris AJ, Bader MF, Frohman MA - J. Cell Biol. (2003)

The isolated PLD1 PH domain associates only weakly with internal and plasma membranes, both in resting cells and stimulated cells. COS-7 cells were transfected with plasmids expressing the PH domain flanked by leader and trailing sequences.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172799&req=5

fig7: The isolated PLD1 PH domain associates only weakly with internal and plasma membranes, both in resting cells and stimulated cells. COS-7 cells were transfected with plasmids expressing the PH domain flanked by leader and trailing sequences.
Mentions: The isolated PLD1 PH domain associates with membranes weakly (Fig. 7). It localizes in resting cells to perinuclear membrane structures. With PMA, a very weak translocation is observed to the PM (Fig. 7), suggesting that the PH domain may contribute to a small degree to regulated translocation, although this is minor at best in the context of the full-length protein (as shown in Fig. 3).

Bottom Line: The PH domain drives PLD1 entry into lipid rafts, which we show to be a step critical for internalization.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University Medical Center, State University of New York at Stony Brook, Stony Brook, NY 11794, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus