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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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Summary of the PLD1 constructs used in this paper. The constructs were NH2-terminally HA- or EGFP-tagged as indicated. See Results for details.
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fig2: Summary of the PLD1 constructs used in this paper. The constructs were NH2-terminally HA- or EGFP-tagged as indicated. See Results for details.

Mentions: To delineate the mechanisms through which PLD1 becomes targeted to different subcellular sites, we generated a series of mutant alleles (Fig. 2). We focused on the four regions most likely to mediate membrane association and specific subcellular targeting: a PX domain, a PH domain, a PI4,5P2 binding motif, and the loop region of PLD1. The mutants were confirmed to be expressed at similar levels and at the expected sizes (unpublished data), and except for the isolated domains, were assayed for regulated activity in vitro using ARF1 as a stimulator. With one exception (PLD1 R691,695G, in which the PI4,5P2-binding site is disrupted), all of the mutants were active at approximately wild-type levels. It should be noted that it is relatively easy to destabilize PLD1 through manipulations involving mutagenesis, resulting in inactive, presumably substantially misfolded, and mislocalized protein (Zhang et al., 1999; Du et al., 2000). A number of variations on the constructs shown in Fig. 2 were inactive and were discarded. Perhaps not surprisingly, the exact boundaries at which deletions were made turned out to be crucial to preserve enzymatic activity (and presumably, adequate folding). This issue could account for discrepancies between our results and a report on mutagenesis of the PLD1 PH domain in which the described mutations generated inactive alleles (Hodgkin et al., 2000).


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)

Summary of the PLD1 constructs used in this paper. The constructs were NH2-terminally HA- or EGFP-tagged as indicated. See Results for details.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Summary of the PLD1 constructs used in this paper. The constructs were NH2-terminally HA- or EGFP-tagged as indicated. See Results for details.
Mentions: To delineate the mechanisms through which PLD1 becomes targeted to different subcellular sites, we generated a series of mutant alleles (Fig. 2). We focused on the four regions most likely to mediate membrane association and specific subcellular targeting: a PX domain, a PH domain, a PI4,5P2 binding motif, and the loop region of PLD1. The mutants were confirmed to be expressed at similar levels and at the expected sizes (unpublished data), and except for the isolated domains, were assayed for regulated activity in vitro using ARF1 as a stimulator. With one exception (PLD1 R691,695G, in which the PI4,5P2-binding site is disrupted), all of the mutants were active at approximately wild-type levels. It should be noted that it is relatively easy to destabilize PLD1 through manipulations involving mutagenesis, resulting in inactive, presumably substantially misfolded, and mislocalized protein (Zhang et al., 1999; Du et al., 2000). A number of variations on the constructs shown in Fig. 2 were inactive and were discarded. Perhaps not surprisingly, the exact boundaries at which deletions were made turned out to be crucial to preserve enzymatic activity (and presumably, adequate folding). This issue could account for discrepancies between our results and a report on mutagenesis of the PLD1 PH domain in which the described mutations generated inactive alleles (Hodgkin et al., 2000).

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