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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 NH2 terminus contains targeting signals for the endosomes and Golgi and is required for internalization, whereas the PI4,5P2-interacting motif mediates recruitment to the PM. (A and C) 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. In C, two examples are shown for each time point. (B) Characterization of the PLD1 PI4,5P2-binding motif. Wild-type (WT) and R691G,R695G (RG) mutant PLD1 proteins purified from baculovirus-infected sf9 cells were mixed with different amounts of liposomes containing PC, PE, and 5% PIP or 5% PI4,5P2. The vesicles were sedimented by centrifugation and the pellets were analyzed by Western blotting for cosedimentation of PLD1.
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fig3: The NH2 terminus contains targeting signals for the endosomes and Golgi and is required for internalization, whereas the PI4,5P2-interacting motif mediates recruitment to the PM. (A and C) 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. In C, two examples are shown for each time point. (B) Characterization of the PLD1 PI4,5P2-binding motif. Wild-type (WT) and R691G,R695G (RG) mutant PLD1 proteins purified from baculovirus-infected sf9 cells were mixed with different amounts of liposomes containing PC, PE, and 5% PIP or 5% PI4,5P2. The vesicles were sedimented by centrifugation and the pellets were analyzed by Western blotting for cosedimentation of PLD1.

Mentions: The PLD1 NH2 terminus would be expected to play a role in localization because it contains both PX and PH domains, each of which have been shown to target many other proteins to membranes through binding to lipid or protein targets. Indeed, a PLD1 allele lacking the PX- and PH-containing NH2 terminus (PLD1-ΔN) that is enzymatically active (Sung et al., 1999b) is cytosolic in quiescent cells (Fig. 3 A), demonstrating that the NH2 terminus is required for localization to perinuclear membrane vesicles. However, on stimulation by PMA, dramatic recruitment to the PM was observed (in >85% of the cells). Moreover, once recruited to the PM, this mutant allele persistently localized there; no reentry into the cell was observed by 4 h after stimulation (Fig. 1, D and E). The first result indicates that the mechanism responsible for PM recruitment does not involve the PX or PH domain, leaving the potential PI4,5P2-interacting site as the most likely candidate. The second result suggests that the PX or PH domain mediates internalization.


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 NH2 terminus contains targeting signals for the endosomes and Golgi and is required for internalization, whereas the PI4,5P2-interacting motif mediates recruitment to the PM. (A and C) 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. In C, two examples are shown for each time point. (B) Characterization of the PLD1 PI4,5P2-binding motif. Wild-type (WT) and R691G,R695G (RG) mutant PLD1 proteins purified from baculovirus-infected sf9 cells were mixed with different amounts of liposomes containing PC, PE, and 5% PIP or 5% PI4,5P2. The vesicles were sedimented by centrifugation and the pellets were analyzed by Western blotting for cosedimentation of PLD1.
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Related In: Results  -  Collection

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fig3: The NH2 terminus contains targeting signals for the endosomes and Golgi and is required for internalization, whereas the PI4,5P2-interacting motif mediates recruitment to the PM. (A and C) 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. In C, two examples are shown for each time point. (B) Characterization of the PLD1 PI4,5P2-binding motif. Wild-type (WT) and R691G,R695G (RG) mutant PLD1 proteins purified from baculovirus-infected sf9 cells were mixed with different amounts of liposomes containing PC, PE, and 5% PIP or 5% PI4,5P2. The vesicles were sedimented by centrifugation and the pellets were analyzed by Western blotting for cosedimentation of PLD1.
Mentions: The PLD1 NH2 terminus would be expected to play a role in localization because it contains both PX and PH domains, each of which have been shown to target many other proteins to membranes through binding to lipid or protein targets. Indeed, a PLD1 allele lacking the PX- and PH-containing NH2 terminus (PLD1-ΔN) that is enzymatically active (Sung et al., 1999b) is cytosolic in quiescent cells (Fig. 3 A), demonstrating that the NH2 terminus is required for localization to perinuclear membrane vesicles. However, on stimulation by PMA, dramatic recruitment to the PM was observed (in >85% of the cells). Moreover, once recruited to the PM, this mutant allele persistently localized there; no reentry into the cell was observed by 4 h after stimulation (Fig. 1, D and E). The first result indicates that the mechanism responsible for PM recruitment does not involve the PX or PH domain, leaving the potential PI4,5P2-interacting site as the most likely candidate. The second result suggests that the PX or PH domain mediates internalization.

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