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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 PX domain mediates endosomal association independent of PI3 kinase products or lipid interactions, but may facilitate reentry through interaction with PI5P. (A and B) COS-7 cells were transiently transfected with wild-type or mutated full-length PLD1 or isolated PX domain constructs as shown in Fig. 2, and were processed as described in Fig. 1. (A) The isolated PLD1 PX domain localizes to cytosolic vesicles, but does not colocalize with EEA1 endosomes (or GM130, not depicted). Not depicted are similar results that were observed for the isolated domain and for the full NH2 terminus (1–212, which contains an extra 70 amino acids, as shown in Fig. 2). (C) COS-7 cells transiently transfected with p40 or PLD1 EGFP-PX domain constructs were followed over time after the addition of varying concentrations of wortmannin. (D) Purified bacterial Nus protein or Nus–PLD1 PX domain fusion protein was used to probe a set of phospholipids spotted on nitrocellulose strips, after which the proteins were detected using an anti-Nus mAb.
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fig5: The PX domain mediates endosomal association independent of PI3 kinase products or lipid interactions, but may facilitate reentry through interaction with PI5P. (A and B) COS-7 cells were transiently transfected with wild-type or mutated full-length PLD1 or isolated PX domain constructs as shown in Fig. 2, and were processed as described in Fig. 1. (A) The isolated PLD1 PX domain localizes to cytosolic vesicles, but does not colocalize with EEA1 endosomes (or GM130, not depicted). Not depicted are similar results that were observed for the isolated domain and for the full NH2 terminus (1–212, which contains an extra 70 amino acids, as shown in Fig. 2). (C) COS-7 cells transiently transfected with p40 or PLD1 EGFP-PX domain constructs were followed over time after the addition of varying concentrations of wortmannin. (D) Purified bacterial Nus protein or Nus–PLD1 PX domain fusion protein was used to probe a set of phospholipids spotted on nitrocellulose strips, after which the proteins were detected using an anti-Nus mAb.

Mentions: Classical PX domains such as p40 bind PI3P, and through this interaction localize to EEA1-containing endosomes (Kanai et al., 2001). This mechanism can be demonstrated in NIH3T3 cells using p40PX-EGFP, which localizes to endosomes in a discrete punctate pattern that is disrupted on addition of wortmannin, a specific inhibitor of PI3 kinases (Fig. 5 C). The isolated PLD1-PX domain localizes to cytosolic vesicles (Fig. 5 A), but colocalization with EEA1 was not observed, suggesting that interaction with PI3P does not underlie its targeting mechanism. Supporting this observation, addition of wortmannin to the transfected cells did not affect PLD1-PX domain localization (Fig. 5 C). Moreover, mutation of R118 did not alter localization in quiescent cells of either the isolated domain or the full-length protein (Fig. 5 B), suggesting that the targeting mechanism here is distinct from the potential phosphoinositide-interacting one that regulates PLD1 reentry (Fig. 4 B). Finally, examination of the binding specificity of the PLD1-PX domain expressed and purified from bacteria revealed a significant interaction with PI5P (Fig. 5 D), a PI that has recently been proposed to accumulate on endocytic vesicles from the hydrolysis of PI4,5P2 (Terebiznik et al., 2002).


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 mediates endosomal association independent of PI3 kinase products or lipid interactions, but may facilitate reentry through interaction with PI5P. (A and B) COS-7 cells were transiently transfected with wild-type or mutated full-length PLD1 or isolated PX domain constructs as shown in Fig. 2, and were processed as described in Fig. 1. (A) The isolated PLD1 PX domain localizes to cytosolic vesicles, but does not colocalize with EEA1 endosomes (or GM130, not depicted). Not depicted are similar results that were observed for the isolated domain and for the full NH2 terminus (1–212, which contains an extra 70 amino acids, as shown in Fig. 2). (C) COS-7 cells transiently transfected with p40 or PLD1 EGFP-PX domain constructs were followed over time after the addition of varying concentrations of wortmannin. (D) Purified bacterial Nus protein or Nus–PLD1 PX domain fusion protein was used to probe a set of phospholipids spotted on nitrocellulose strips, after which the proteins were detected using an anti-Nus mAb.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172799&req=5

fig5: The PX domain mediates endosomal association independent of PI3 kinase products or lipid interactions, but may facilitate reentry through interaction with PI5P. (A and B) COS-7 cells were transiently transfected with wild-type or mutated full-length PLD1 or isolated PX domain constructs as shown in Fig. 2, and were processed as described in Fig. 1. (A) The isolated PLD1 PX domain localizes to cytosolic vesicles, but does not colocalize with EEA1 endosomes (or GM130, not depicted). Not depicted are similar results that were observed for the isolated domain and for the full NH2 terminus (1–212, which contains an extra 70 amino acids, as shown in Fig. 2). (C) COS-7 cells transiently transfected with p40 or PLD1 EGFP-PX domain constructs were followed over time after the addition of varying concentrations of wortmannin. (D) Purified bacterial Nus protein or Nus–PLD1 PX domain fusion protein was used to probe a set of phospholipids spotted on nitrocellulose strips, after which the proteins were detected using an anti-Nus mAb.
Mentions: Classical PX domains such as p40 bind PI3P, and through this interaction localize to EEA1-containing endosomes (Kanai et al., 2001). This mechanism can be demonstrated in NIH3T3 cells using p40PX-EGFP, which localizes to endosomes in a discrete punctate pattern that is disrupted on addition of wortmannin, a specific inhibitor of PI3 kinases (Fig. 5 C). The isolated PLD1-PX domain localizes to cytosolic vesicles (Fig. 5 A), but colocalization with EEA1 was not observed, suggesting that interaction with PI3P does not underlie its targeting mechanism. Supporting this observation, addition of wortmannin to the transfected cells did not affect PLD1-PX domain localization (Fig. 5 C). Moreover, mutation of R118 did not alter localization in quiescent cells of either the isolated domain or the full-length protein (Fig. 5 B), suggesting that the targeting mechanism here is distinct from the potential phosphoinositide-interacting one that regulates PLD1 reentry (Fig. 4 B). Finally, examination of the binding specificity of the PLD1-PX domain expressed and purified from bacteria revealed a significant interaction with PI5P (Fig. 5 D), a PI that has recently been proposed to accumulate on endocytic vesicles from the hydrolysis of PI4,5P2 (Terebiznik et al., 2002).

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