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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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A model for regulated cycling of PLD1. See Discussion for details. In brief, in quiescent COS-7 cells, PLD1 localizes to a complex set of perinuclear and cytosolic vesicles (top schema, heavy black circle). This is mediated by a non-PI interaction by the PX domain, the palmitoylated PH domain, and weak interactions with PI4,5P2 by the central basic amino acid–rich motif, although the different domains most likely preferentially target distinct subpopulations of vesicles. In PC12 cells, a combination of PX domain interactions and interactions with PI4,5P2 suffice to recruit PLD1 to the PM (bottom schema, heavy black circle). With higher levels of stimulation, such as exposure to PMA for COS-7 cells, the PI4,5P2 interaction alone suffices to promote PM association. However, to reenter the cell with normal kinetics, PLD1 needs to enter into lipid rafts, and for this, the PH domain to be palmitoylated. Once in rafts, the PX domain association with the PM ceases; but a new PI-dependent interaction takes place, potentially through the binding of PX to PI5P, which facilitates translocation of PLD1 to vesicles internalizing through endocytosis. In PC12 cells, secretagogue-evoked stimulation (depolarization) recruits PLD1 into sites of active exocytosis, which are found in rafts and are marked by increased levels of PI4,5P2. Palmitoylation of the PH domain is similarly required for this recruitment, but once it happens, the PX domain interaction is no longer required for membrane association or for PLD1 functional promotion of exocytosis. How PLD1 returns to its original location remains to be determined.
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fig9: A model for regulated cycling of PLD1. See Discussion for details. In brief, in quiescent COS-7 cells, PLD1 localizes to a complex set of perinuclear and cytosolic vesicles (top schema, heavy black circle). This is mediated by a non-PI interaction by the PX domain, the palmitoylated PH domain, and weak interactions with PI4,5P2 by the central basic amino acid–rich motif, although the different domains most likely preferentially target distinct subpopulations of vesicles. In PC12 cells, a combination of PX domain interactions and interactions with PI4,5P2 suffice to recruit PLD1 to the PM (bottom schema, heavy black circle). With higher levels of stimulation, such as exposure to PMA for COS-7 cells, the PI4,5P2 interaction alone suffices to promote PM association. However, to reenter the cell with normal kinetics, PLD1 needs to enter into lipid rafts, and for this, the PH domain to be palmitoylated. Once in rafts, the PX domain association with the PM ceases; but a new PI-dependent interaction takes place, potentially through the binding of PX to PI5P, which facilitates translocation of PLD1 to vesicles internalizing through endocytosis. In PC12 cells, secretagogue-evoked stimulation (depolarization) recruits PLD1 into sites of active exocytosis, which are found in rafts and are marked by increased levels of PI4,5P2. Palmitoylation of the PH domain is similarly required for this recruitment, but once it happens, the PX domain interaction is no longer required for membrane association or for PLD1 functional promotion of exocytosis. How PLD1 returns to its original location remains to be determined.

Mentions: The cycling steps we propose for PLD1 are summarized in Fig. 9. Key observations were similar in COS-7 and PC12, although there were some differences as well. Whether this reflects the different culture and stimulatory conditions used or whether it indicates that the PX, PH, and PI4,5P2 associations combine to play out somewhat differently in different cell types remains to be determined. Further clarification and extension of the proposed model will come from identifying the relevant protein and/or lipid targets with which PLD1 interacts, and from following PLD1 trafficking in real time from selected compartments using photoactivatable alleles.


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)

A model for regulated cycling of PLD1. See Discussion for details. In brief, in quiescent COS-7 cells, PLD1 localizes to a complex set of perinuclear and cytosolic vesicles (top schema, heavy black circle). This is mediated by a non-PI interaction by the PX domain, the palmitoylated PH domain, and weak interactions with PI4,5P2 by the central basic amino acid–rich motif, although the different domains most likely preferentially target distinct subpopulations of vesicles. In PC12 cells, a combination of PX domain interactions and interactions with PI4,5P2 suffice to recruit PLD1 to the PM (bottom schema, heavy black circle). With higher levels of stimulation, such as exposure to PMA for COS-7 cells, the PI4,5P2 interaction alone suffices to promote PM association. However, to reenter the cell with normal kinetics, PLD1 needs to enter into lipid rafts, and for this, the PH domain to be palmitoylated. Once in rafts, the PX domain association with the PM ceases; but a new PI-dependent interaction takes place, potentially through the binding of PX to PI5P, which facilitates translocation of PLD1 to vesicles internalizing through endocytosis. In PC12 cells, secretagogue-evoked stimulation (depolarization) recruits PLD1 into sites of active exocytosis, which are found in rafts and are marked by increased levels of PI4,5P2. Palmitoylation of the PH domain is similarly required for this recruitment, but once it happens, the PX domain interaction is no longer required for membrane association or for PLD1 functional promotion of exocytosis. How PLD1 returns to its original location remains to be determined.
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Related In: Results  -  Collection

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fig9: A model for regulated cycling of PLD1. See Discussion for details. In brief, in quiescent COS-7 cells, PLD1 localizes to a complex set of perinuclear and cytosolic vesicles (top schema, heavy black circle). This is mediated by a non-PI interaction by the PX domain, the palmitoylated PH domain, and weak interactions with PI4,5P2 by the central basic amino acid–rich motif, although the different domains most likely preferentially target distinct subpopulations of vesicles. In PC12 cells, a combination of PX domain interactions and interactions with PI4,5P2 suffice to recruit PLD1 to the PM (bottom schema, heavy black circle). With higher levels of stimulation, such as exposure to PMA for COS-7 cells, the PI4,5P2 interaction alone suffices to promote PM association. However, to reenter the cell with normal kinetics, PLD1 needs to enter into lipid rafts, and for this, the PH domain to be palmitoylated. Once in rafts, the PX domain association with the PM ceases; but a new PI-dependent interaction takes place, potentially through the binding of PX to PI5P, which facilitates translocation of PLD1 to vesicles internalizing through endocytosis. In PC12 cells, secretagogue-evoked stimulation (depolarization) recruits PLD1 into sites of active exocytosis, which are found in rafts and are marked by increased levels of PI4,5P2. Palmitoylation of the PH domain is similarly required for this recruitment, but once it happens, the PX domain interaction is no longer required for membrane association or for PLD1 functional promotion of exocytosis. How PLD1 returns to its original location remains to be determined.
Mentions: The cycling steps we propose for PLD1 are summarized in Fig. 9. Key observations were similar in COS-7 and PC12, although there were some differences as well. Whether this reflects the different culture and stimulatory conditions used or whether it indicates that the PX, PH, and PI4,5P2 associations combine to play out somewhat differently in different cell types remains to be determined. Further clarification and extension of the proposed model will come from identifying the relevant protein and/or lipid targets with which PLD1 interacts, and from following PLD1 trafficking in real time from selected compartments using photoactivatable alleles.

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