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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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Related in: MedlinePlus

The PX domain is required for PLD1 internalization potentially through phosphoinositide interactions, but is not required for localization to at least some types of endosomes. 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. Two examples are shown for each time point to indicate how cells were scored.
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fig4: The PX domain is required for PLD1 internalization potentially through phosphoinositide interactions, but is not required for localization to at least some types of endosomes. 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. Two examples are shown for each time point to indicate how cells were scored.

Mentions: The PX domain plays a relatively subtle role in PLD1 localization in serum-starved cells (Fig. 4 A) because an allele lacking the PX domain is still found in perinuclear vesicles. However, the nature of the vesicles targeted appears to be shifted because nearly uniform colocalization in sorting/recycling endosomes was observed with the TfR and the dominant-negative ARF6 mutant T27N (unpublished data; pattern of localization of TfR and ARF6-T27N discussed in D'Souza et al., 1995), but colocalization with GM130 or EEA1 was decreased (unpublished data). However, more obviously, the PLD1-ΔPX allele still underwent robust translocation to the PM after PMA stimulation, but did not undergo wild-type-like reentry (Fig. 4 A). Similar results were observed for point mutants (R118, F120/R179) in the PX domain at amino acid residues predicted to be required for interaction with phosphoinositide lipid anchors (based on the published structures of the p40 and p47 PX domains and sequence alignment of the entire set of known PX domains; for review see Sato et al., 2001). Together, these findings suggest that the PX domain is required in a phosphoinositide-dependent process for PLD1 to reenter COS-7 cells once it has become localized to the PM.


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 PX domain is required for PLD1 internalization potentially through phosphoinositide interactions, but is not required for localization to at least some types of endosomes. 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. Two examples are shown for each time point to indicate how cells were scored.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: The PX domain is required for PLD1 internalization potentially through phosphoinositide interactions, but is not required for localization to at least some types of endosomes. 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. Two examples are shown for each time point to indicate how cells were scored.
Mentions: The PX domain plays a relatively subtle role in PLD1 localization in serum-starved cells (Fig. 4 A) because an allele lacking the PX domain is still found in perinuclear vesicles. However, the nature of the vesicles targeted appears to be shifted because nearly uniform colocalization in sorting/recycling endosomes was observed with the TfR and the dominant-negative ARF6 mutant T27N (unpublished data; pattern of localization of TfR and ARF6-T27N discussed in D'Souza et al., 1995), but colocalization with GM130 or EEA1 was decreased (unpublished data). However, more obviously, the PLD1-ΔPX allele still underwent robust translocation to the PM after PMA stimulation, but did not undergo wild-type-like reentry (Fig. 4 A). Similar results were observed for point mutants (R118, F120/R179) in the PX domain at amino acid residues predicted to be required for interaction with phosphoinositide lipid anchors (based on the published structures of the p40 and p47 PX domains and sequence alignment of the entire set of known PX domains; for review see Sato et al., 2001). Together, these findings suggest that the PX domain is required in a phosphoinositide-dependent process for PLD1 to reenter COS-7 cells once it has become localized to the PM.

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