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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 PLD1 PH domain targets endosomes in quiescent cells and mediates internalization from the PM through controlling entry into lipid rafts. COS-7 cells were transiently transfected with deletion or mutated PLD1 constructs as shown in Fig. 2, and were processed as described in Fig. 1. (A and B) Two examples are shown for the deletion construct at each time point. Where indicated (rightmost quantification panel), methyl-β-cyclodextrin was added to the culture medium for 45 min and then thoroughly washed away before PMA stimulation.
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fig6: The PLD1 PH domain targets endosomes in quiescent cells and mediates internalization from the PM through controlling entry into lipid rafts. COS-7 cells were transiently transfected with deletion or mutated PLD1 constructs as shown in Fig. 2, and were processed as described in Fig. 1. (A and B) Two examples are shown for the deletion construct at each time point. Where indicated (rightmost quantification panel), methyl-β-cyclodextrin was added to the culture medium for 45 min and then thoroughly washed away before PMA stimulation.

Mentions: A catalytically active PLD1-ΔPH allele exhibited cytosolic localization (Fig. 6, A and B) and exhibited some (but not complete) PMA-stimulated translocation to the PM followed by delayed reentry. The lack of robust PMA-elicited translocation (for example, in comparison to the PLD1-ΔN allele described in Fig. 3, which lacks even more sequence) suggests that the deletion of the PH domain may have affected the functionality of the other domains, despite the fact that catalytic activity was maintained.


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 PLD1 PH domain targets endosomes in quiescent cells and mediates internalization from the PM through controlling entry into lipid rafts. COS-7 cells were transiently transfected with deletion or mutated PLD1 constructs as shown in Fig. 2, and were processed as described in Fig. 1. (A and B) Two examples are shown for the deletion construct at each time point. Where indicated (rightmost quantification panel), methyl-β-cyclodextrin was added to the culture medium for 45 min and then thoroughly washed away before PMA stimulation.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: The PLD1 PH domain targets endosomes in quiescent cells and mediates internalization from the PM through controlling entry into lipid rafts. COS-7 cells were transiently transfected with deletion or mutated PLD1 constructs as shown in Fig. 2, and were processed as described in Fig. 1. (A and B) Two examples are shown for the deletion construct at each time point. Where indicated (rightmost quantification panel), methyl-β-cyclodextrin was added to the culture medium for 45 min and then thoroughly washed away before PMA stimulation.
Mentions: A catalytically active PLD1-ΔPH allele exhibited cytosolic localization (Fig. 6, A and B) and exhibited some (but not complete) PMA-stimulated translocation to the PM followed by delayed reentry. The lack of robust PMA-elicited translocation (for example, in comparison to the PLD1-ΔN allele described in Fig. 3, which lacks even more sequence) suggests that the deletion of the PH domain may have affected the functionality of the other domains, despite the fact that catalytic activity was maintained.

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