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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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PLD1 recycles between PM and intracellular vesicles in an activity-facilitated manner on PMA stimulation. COS-7 cells were transiently transfected with an HA-tagged PLD1 expression plasmid. 36 h later, the cells were stimulated with PMA for varying periods of time (A, C, and D) and then fixed and immunostained using an anti-HA mAb. Images were captured using a confocal microscope (TCS SP2; Leica) using a green fluorophore-labeled secondary antibody. Colocalization with EEA1, GM130, and TfR (B) was performed using far-red as the second fluorophore. On stimulation by PMA, PLD1 translocates to the PM; shown is a 2-h time point (C). C′ and D′ show additional images of cells in which translocation or return was not complete at the respective time points. E depicts a tabulation of percent localization for 300 cells at each time point, including SDs that were determined by conducting the experiment three times. Vesicles (V), plasma membrane (PM), and intermediate cells (PM/V) were scored as indicated by the labels in the bottom right corner of each image.
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fig1: PLD1 recycles between PM and intracellular vesicles in an activity-facilitated manner on PMA stimulation. COS-7 cells were transiently transfected with an HA-tagged PLD1 expression plasmid. 36 h later, the cells were stimulated with PMA for varying periods of time (A, C, and D) and then fixed and immunostained using an anti-HA mAb. Images were captured using a confocal microscope (TCS SP2; Leica) using a green fluorophore-labeled secondary antibody. Colocalization with EEA1, GM130, and TfR (B) was performed using far-red as the second fluorophore. On stimulation by PMA, PLD1 translocates to the PM; shown is a 2-h time point (C). C′ and D′ show additional images of cells in which translocation or return was not complete at the respective time points. E depicts a tabulation of percent localization for 300 cells at each time point, including SDs that were determined by conducting the experiment three times. Vesicles (V), plasma membrane (PM), and intermediate cells (PM/V) were scored as indicated by the labels in the bottom right corner of each image.

Mentions: A typical perinuclear vesicular localization pattern for PLD1 in resting cells as reported previously by us and others (Freyberg et al., 2001; Lucocq et al., 2001) is shown in Fig. 1 A. A subset of the vesicles colocalize with the transferrin receptor (TfR), which is found in sorting/recycling endosomes (Fig. 1 B); others colocalize with EEA1, an early endosomal marker, and some colocalization is also observed with GM130, a Golgi marker. Infrequently (unpublished data), a small amount of PLD1 is found on the PM. The relative distribution of wild-type PLD1 in serum-starved cells was determined by examining 300 cells. As shown in the left panel of Fig. 1 E, PLD1 was exclusively found in such perinuclear vesicles in >90% of the cells. PMA stimulation, which activates PKC, resulted in translocation of most or all of the PLD1 to the PM and peripheral vesicles (Fig. 1, C and C′) in the majority of cells (>80%, Fig. 1 E) within 2 h. By 4 h after stimulation, much of the PLD1 had returned to the perinuclear vesicles (Fig. 1, D and E), although some residual localization on the PM remained in some cells (Fig. 1 D′).


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)

PLD1 recycles between PM and intracellular vesicles in an activity-facilitated manner on PMA stimulation. COS-7 cells were transiently transfected with an HA-tagged PLD1 expression plasmid. 36 h later, the cells were stimulated with PMA for varying periods of time (A, C, and D) and then fixed and immunostained using an anti-HA mAb. Images were captured using a confocal microscope (TCS SP2; Leica) using a green fluorophore-labeled secondary antibody. Colocalization with EEA1, GM130, and TfR (B) was performed using far-red as the second fluorophore. On stimulation by PMA, PLD1 translocates to the PM; shown is a 2-h time point (C). C′ and D′ show additional images of cells in which translocation or return was not complete at the respective time points. E depicts a tabulation of percent localization for 300 cells at each time point, including SDs that were determined by conducting the experiment three times. Vesicles (V), plasma membrane (PM), and intermediate cells (PM/V) were scored as indicated by the labels in the bottom right corner of each image.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172799&req=5

fig1: PLD1 recycles between PM and intracellular vesicles in an activity-facilitated manner on PMA stimulation. COS-7 cells were transiently transfected with an HA-tagged PLD1 expression plasmid. 36 h later, the cells were stimulated with PMA for varying periods of time (A, C, and D) and then fixed and immunostained using an anti-HA mAb. Images were captured using a confocal microscope (TCS SP2; Leica) using a green fluorophore-labeled secondary antibody. Colocalization with EEA1, GM130, and TfR (B) was performed using far-red as the second fluorophore. On stimulation by PMA, PLD1 translocates to the PM; shown is a 2-h time point (C). C′ and D′ show additional images of cells in which translocation or return was not complete at the respective time points. E depicts a tabulation of percent localization for 300 cells at each time point, including SDs that were determined by conducting the experiment three times. Vesicles (V), plasma membrane (PM), and intermediate cells (PM/V) were scored as indicated by the labels in the bottom right corner of each image.
Mentions: A typical perinuclear vesicular localization pattern for PLD1 in resting cells as reported previously by us and others (Freyberg et al., 2001; Lucocq et al., 2001) is shown in Fig. 1 A. A subset of the vesicles colocalize with the transferrin receptor (TfR), which is found in sorting/recycling endosomes (Fig. 1 B); others colocalize with EEA1, an early endosomal marker, and some colocalization is also observed with GM130, a Golgi marker. Infrequently (unpublished data), a small amount of PLD1 is found on the PM. The relative distribution of wild-type PLD1 in serum-starved cells was determined by examining 300 cells. As shown in the left panel of Fig. 1 E, PLD1 was exclusively found in such perinuclear vesicles in >90% of the cells. PMA stimulation, which activates PKC, resulted in translocation of most or all of the PLD1 to the PM and peripheral vesicles (Fig. 1, C and C′) in the majority of cells (>80%, Fig. 1 E) within 2 h. By 4 h after stimulation, much of the PLD1 had returned to the perinuclear vesicles (Fig. 1, D and E), although some residual localization on the PM remained in some cells (Fig. 1 D′).

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