Limits...
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.

Show MeSH

Related in: MedlinePlus

PLD1 localization affects regulated exocytosis in PC12 cells. PC12 cells were cotransfected with plasmids encoding hGH and tagged PLD1 alleles. 48 h after transfection, cells were incubated for 10 min in calcium-free Locke's solution (nondepolarized) or in 59 mM K+ solution (depolarized). (Top) Cells were immunostained to visualize the PLD1 protein by confocal microscopy. Bar, 5 μm. (Bottom) Assay for regulated exocytosis. The total GH content in control and PLD1-expressing cells was in the range of 5 ng/well. The net K+-evoked secretory response was obtained by subtracting the basal hGH release from the hGH release evoked by 59 mM K+. In these experiments, basal release ranged from 5.0 to 5.7%. The net K+-evoked GH release was 25.7% ± 0.4 from empty plasmid-transfected control cells, and, depending on the allele, ranged from 12.8 to 32.3% in PLD1-expressing cells. Finally, the net secretion under experimental conditions was divided by the net secretion in control cultures to estimate the percent change in regulated exocytosis. The experiments were performed twice in triplicate, and SEM is presented.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172799&req=5

fig8: PLD1 localization affects regulated exocytosis in PC12 cells. PC12 cells were cotransfected with plasmids encoding hGH and tagged PLD1 alleles. 48 h after transfection, cells were incubated for 10 min in calcium-free Locke's solution (nondepolarized) or in 59 mM K+ solution (depolarized). (Top) Cells were immunostained to visualize the PLD1 protein by confocal microscopy. Bar, 5 μm. (Bottom) Assay for regulated exocytosis. The total GH content in control and PLD1-expressing cells was in the range of 5 ng/well. The net K+-evoked secretory response was obtained by subtracting the basal hGH release from the hGH release evoked by 59 mM K+. In these experiments, basal release ranged from 5.0 to 5.7%. The net K+-evoked GH release was 25.7% ± 0.4 from empty plasmid-transfected control cells, and, depending on the allele, ranged from 12.8 to 32.3% in PLD1-expressing cells. Finally, the net secretion under experimental conditions was divided by the net secretion in control cultures to estimate the percent change in regulated exocytosis. The experiments were performed twice in triplicate, and SEM is presented.

Mentions: Previously, we reported that PLD1 is expressed in neuroendocrine cells and facilitates regulated exocytosis of secretory granules (Vitale et al., 2001). In the assay system used for those analyses, the PC12 cells used were cultured in 15% serum, and exocytosis was triggered by the elevation of potassium, which induces depolarization. PLD1 localized to the PM in these cells and increased exocytotic responses when overexpressed, whereas the inactive allele, PLD1-K898R, which localized similarly, inhibited exocytosis (Fig. 8; see also Vitale et al., 2001). We also reported that interaction with PI4,5P2 was potentially required for the PM localization in nondepolarized cells because the PLD1-R691,695G mutant allele localized to the cytosol and did not alter the extent of exocytosis. Using some of the mutant alleles generated in this report, we now further examine in PC12 cells whether the different PLD1 membrane-association motifs control its subcellular distribution, and what the importance of PLD1 localization is in regulating exocytosis.


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 localization affects regulated exocytosis in PC12 cells. PC12 cells were cotransfected with plasmids encoding hGH and tagged PLD1 alleles. 48 h after transfection, cells were incubated for 10 min in calcium-free Locke's solution (nondepolarized) or in 59 mM K+ solution (depolarized). (Top) Cells were immunostained to visualize the PLD1 protein by confocal microscopy. Bar, 5 μm. (Bottom) Assay for regulated exocytosis. The total GH content in control and PLD1-expressing cells was in the range of 5 ng/well. The net K+-evoked secretory response was obtained by subtracting the basal hGH release from the hGH release evoked by 59 mM K+. In these experiments, basal release ranged from 5.0 to 5.7%. The net K+-evoked GH release was 25.7% ± 0.4 from empty plasmid-transfected control cells, and, depending on the allele, ranged from 12.8 to 32.3% in PLD1-expressing cells. Finally, the net secretion under experimental conditions was divided by the net secretion in control cultures to estimate the percent change in regulated exocytosis. The experiments were performed twice in triplicate, and SEM is presented.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: PLD1 localization affects regulated exocytosis in PC12 cells. PC12 cells were cotransfected with plasmids encoding hGH and tagged PLD1 alleles. 48 h after transfection, cells were incubated for 10 min in calcium-free Locke's solution (nondepolarized) or in 59 mM K+ solution (depolarized). (Top) Cells were immunostained to visualize the PLD1 protein by confocal microscopy. Bar, 5 μm. (Bottom) Assay for regulated exocytosis. The total GH content in control and PLD1-expressing cells was in the range of 5 ng/well. The net K+-evoked secretory response was obtained by subtracting the basal hGH release from the hGH release evoked by 59 mM K+. In these experiments, basal release ranged from 5.0 to 5.7%. The net K+-evoked GH release was 25.7% ± 0.4 from empty plasmid-transfected control cells, and, depending on the allele, ranged from 12.8 to 32.3% in PLD1-expressing cells. Finally, the net secretion under experimental conditions was divided by the net secretion in control cultures to estimate the percent change in regulated exocytosis. The experiments were performed twice in triplicate, and SEM is presented.
Mentions: Previously, we reported that PLD1 is expressed in neuroendocrine cells and facilitates regulated exocytosis of secretory granules (Vitale et al., 2001). In the assay system used for those analyses, the PC12 cells used were cultured in 15% serum, and exocytosis was triggered by the elevation of potassium, which induces depolarization. PLD1 localized to the PM in these cells and increased exocytotic responses when overexpressed, whereas the inactive allele, PLD1-K898R, which localized similarly, inhibited exocytosis (Fig. 8; see also Vitale et al., 2001). We also reported that interaction with PI4,5P2 was potentially required for the PM localization in nondepolarized cells because the PLD1-R691,695G mutant allele localized to the cytosol and did not alter the extent of exocytosis. Using some of the mutant alleles generated in this report, we now further examine in PC12 cells whether the different PLD1 membrane-association motifs control its subcellular distribution, and what the importance of PLD1 localization is in regulating exocytosis.

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