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Phospholipase D stimulates release of nascent secretory vesicles from the trans-Golgi network.

Chen YG, Siddhanta A, Austin CD, Hammond SM, Sung TC, Frohman MA, Morris AJ, Shields D - J. Cell Biol. (1997)

Bottom Line: Phospholipase D (PLD) is a phospholipid hydrolyzing enzyme whose activation has been implicated in mediating signal transduction pathways, cell growth, and membrane trafficking in mammalian cells.Several laboratories have demonstrated that small GTP-binding proteins including ADP-ribosylation factor (ARF) can stimulate PLD activity in vitro and an ARF-activated PLD activity has been found in Golgi membranes.Our results suggest that ARF regulation of PLD activity plays an important role in the release of nascent secretory vesicles from the TGN.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.

ABSTRACT
Phospholipase D (PLD) is a phospholipid hydrolyzing enzyme whose activation has been implicated in mediating signal transduction pathways, cell growth, and membrane trafficking in mammalian cells. Several laboratories have demonstrated that small GTP-binding proteins including ADP-ribosylation factor (ARF) can stimulate PLD activity in vitro and an ARF-activated PLD activity has been found in Golgi membranes. Since ARF-1 has also been shown to enhance release of nascent secretory vesicles from the TGN of endocrine cells, we hypothesized that this reaction occurred via PLD activation. Using a permeabilized cell system derived from growth hormone and prolactin-secreting pituitary GH3 cells, we demonstrate that immunoaffinity-purified human PLD1 stimulated nascent secretory vesicle budding from the TGN approximately twofold. In contrast, a similarly purified but enzymatically inactive mutant form of PLD1, designated Lys898Arg, had no effect on vesicle budding when added to the permeabilized cells. The release of nascent secretory vesicles from the TGN was sensitive to 1% 1-butanol, a concentration that inhibited PLD-catalyzed formation of phosphatidic acid. Furthermore, ARF-1 stimulated endogenous PLD activity in Golgi membranes approximately threefold and this activation correlated with its enhancement of vesicle budding. Our results suggest that ARF regulation of PLD activity plays an important role in the release of nascent secretory vesicles from the TGN.

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Human PLD1 hydrolyzes Golgi membrane  phospholipids. GH3 cells  were incubated for 24 h with  [3H]oleic acid and Golgi  membranes isolated by  floatation on a sucrose gradient (Austin and Shields,  1996). The isolated membranes were incubated with  1 M KCl at 4°C for 10 min to  remove endogenous PLD.  The salt-treated membranes were pelleted by centrifugation at  150,000 g for 50 min and resuspended in incubation buffer. Equal  aliquots of the salt-washed membrane suspension (∼100,000  cpm) were incubated with 0.3% butanol to generate phosphatidylbutanol in the presence of ∼1 μg/ml of the Sf-9 cell extract  expressing human PLD1 (lanes 2–4) or the control extract (lane  5) in a reaction containing (lanes 1, 2, 4, 5) or lacking (lane 3) recombinant myristoylated ARF-1 (m-ARF*). When present,  ARF was used at a final concentration of ∼1 μM and GTPγS at  30 μM.
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Figure 6: Human PLD1 hydrolyzes Golgi membrane phospholipids. GH3 cells were incubated for 24 h with [3H]oleic acid and Golgi membranes isolated by floatation on a sucrose gradient (Austin and Shields, 1996). The isolated membranes were incubated with 1 M KCl at 4°C for 10 min to remove endogenous PLD. The salt-treated membranes were pelleted by centrifugation at 150,000 g for 50 min and resuspended in incubation buffer. Equal aliquots of the salt-washed membrane suspension (∼100,000 cpm) were incubated with 0.3% butanol to generate phosphatidylbutanol in the presence of ∼1 μg/ml of the Sf-9 cell extract expressing human PLD1 (lanes 2–4) or the control extract (lane 5) in a reaction containing (lanes 1, 2, 4, 5) or lacking (lane 3) recombinant myristoylated ARF-1 (m-ARF*). When present, ARF was used at a final concentration of ∼1 μM and GTPγS at 30 μM.

Mentions: The above experiments, using immunopurified native and mutant human PLD1, strongly suggest that PLD catalytic activity mediates vesicle release from the TGN. To determine if exogenously added human PLD hydrolyzed endogenous Golgi membrane phospholipids, GH3 cells were incubated with [3H]oleate to radiolabel phospholipids, Golgi membranes were isolated and treated with 1 M KCl to extract endogenous PLD1 from the membranes (Hammond et al., 1997; Fig. 6). The salt-treated Golgi membranes were incubated with Sf-9 cell extracts under vesicle budding conditions in the presence of 0.3% 1-butanol. The ability of human PLD1 to hydrolyze endogenous phospholipids was then determined by measuring the formation of PtdBut (Fig. 6). Incubation of the Golgi membranes with recombinant myristoylated ARF had no effect on lipid hydrolysis since the endogenous PLD had been effectively removed from the membranes by high salt treatment (Fig. 6, lane 1). Addition of the PLD1 containing Sf-9 extract (Fig. 6, lane 2) but not the control extract (Fig. 6, lane 5) hydrolyzed the phospholipids to generate PtdBut. As previously observed (Brown et al., 1993; Cockcroft et al., 1994; Hammond et al., 1995), PLD activity was enhanced in the presence of GTPγS and myrARF (Fig. 6, lanes 2 and 3). Most importantly, these results demonstrated that human PLD1 was active in hydrolyzing endogenous Golgi membrane phospholipids, and that under these conditions vesicle budding was enhanced.


Phospholipase D stimulates release of nascent secretory vesicles from the trans-Golgi network.

Chen YG, Siddhanta A, Austin CD, Hammond SM, Sung TC, Frohman MA, Morris AJ, Shields D - J. Cell Biol. (1997)

Human PLD1 hydrolyzes Golgi membrane  phospholipids. GH3 cells  were incubated for 24 h with  [3H]oleic acid and Golgi  membranes isolated by  floatation on a sucrose gradient (Austin and Shields,  1996). The isolated membranes were incubated with  1 M KCl at 4°C for 10 min to  remove endogenous PLD.  The salt-treated membranes were pelleted by centrifugation at  150,000 g for 50 min and resuspended in incubation buffer. Equal  aliquots of the salt-washed membrane suspension (∼100,000  cpm) were incubated with 0.3% butanol to generate phosphatidylbutanol in the presence of ∼1 μg/ml of the Sf-9 cell extract  expressing human PLD1 (lanes 2–4) or the control extract (lane  5) in a reaction containing (lanes 1, 2, 4, 5) or lacking (lane 3) recombinant myristoylated ARF-1 (m-ARF*). When present,  ARF was used at a final concentration of ∼1 μM and GTPγS at  30 μM.
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Related In: Results  -  Collection

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Figure 6: Human PLD1 hydrolyzes Golgi membrane phospholipids. GH3 cells were incubated for 24 h with [3H]oleic acid and Golgi membranes isolated by floatation on a sucrose gradient (Austin and Shields, 1996). The isolated membranes were incubated with 1 M KCl at 4°C for 10 min to remove endogenous PLD. The salt-treated membranes were pelleted by centrifugation at 150,000 g for 50 min and resuspended in incubation buffer. Equal aliquots of the salt-washed membrane suspension (∼100,000 cpm) were incubated with 0.3% butanol to generate phosphatidylbutanol in the presence of ∼1 μg/ml of the Sf-9 cell extract expressing human PLD1 (lanes 2–4) or the control extract (lane 5) in a reaction containing (lanes 1, 2, 4, 5) or lacking (lane 3) recombinant myristoylated ARF-1 (m-ARF*). When present, ARF was used at a final concentration of ∼1 μM and GTPγS at 30 μM.
Mentions: The above experiments, using immunopurified native and mutant human PLD1, strongly suggest that PLD catalytic activity mediates vesicle release from the TGN. To determine if exogenously added human PLD hydrolyzed endogenous Golgi membrane phospholipids, GH3 cells were incubated with [3H]oleate to radiolabel phospholipids, Golgi membranes were isolated and treated with 1 M KCl to extract endogenous PLD1 from the membranes (Hammond et al., 1997; Fig. 6). The salt-treated Golgi membranes were incubated with Sf-9 cell extracts under vesicle budding conditions in the presence of 0.3% 1-butanol. The ability of human PLD1 to hydrolyze endogenous phospholipids was then determined by measuring the formation of PtdBut (Fig. 6). Incubation of the Golgi membranes with recombinant myristoylated ARF had no effect on lipid hydrolysis since the endogenous PLD had been effectively removed from the membranes by high salt treatment (Fig. 6, lane 1). Addition of the PLD1 containing Sf-9 extract (Fig. 6, lane 2) but not the control extract (Fig. 6, lane 5) hydrolyzed the phospholipids to generate PtdBut. As previously observed (Brown et al., 1993; Cockcroft et al., 1994; Hammond et al., 1995), PLD activity was enhanced in the presence of GTPγS and myrARF (Fig. 6, lanes 2 and 3). Most importantly, these results demonstrated that human PLD1 was active in hydrolyzing endogenous Golgi membrane phospholipids, and that under these conditions vesicle budding was enhanced.

Bottom Line: Phospholipase D (PLD) is a phospholipid hydrolyzing enzyme whose activation has been implicated in mediating signal transduction pathways, cell growth, and membrane trafficking in mammalian cells.Several laboratories have demonstrated that small GTP-binding proteins including ADP-ribosylation factor (ARF) can stimulate PLD activity in vitro and an ARF-activated PLD activity has been found in Golgi membranes.Our results suggest that ARF regulation of PLD activity plays an important role in the release of nascent secretory vesicles from the TGN.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.

ABSTRACT
Phospholipase D (PLD) is a phospholipid hydrolyzing enzyme whose activation has been implicated in mediating signal transduction pathways, cell growth, and membrane trafficking in mammalian cells. Several laboratories have demonstrated that small GTP-binding proteins including ADP-ribosylation factor (ARF) can stimulate PLD activity in vitro and an ARF-activated PLD activity has been found in Golgi membranes. Since ARF-1 has also been shown to enhance release of nascent secretory vesicles from the TGN of endocrine cells, we hypothesized that this reaction occurred via PLD activation. Using a permeabilized cell system derived from growth hormone and prolactin-secreting pituitary GH3 cells, we demonstrate that immunoaffinity-purified human PLD1 stimulated nascent secretory vesicle budding from the TGN approximately twofold. In contrast, a similarly purified but enzymatically inactive mutant form of PLD1, designated Lys898Arg, had no effect on vesicle budding when added to the permeabilized cells. The release of nascent secretory vesicles from the TGN was sensitive to 1% 1-butanol, a concentration that inhibited PLD-catalyzed formation of phosphatidic acid. Furthermore, ARF-1 stimulated endogenous PLD activity in Golgi membranes approximately threefold and this activation correlated with its enhancement of vesicle budding. Our results suggest that ARF regulation of PLD activity plays an important role in the release of nascent secretory vesicles from the TGN.

Show MeSH
Related in: MedlinePlus