Intracellular Localization of Phospholipase D1 in Mammalian Cells
01 Apr 2001-Molecular Biology of the Cell (American Society for Cell Biology)-Vol. 12, Iss: 4, pp 943-955
TL;DR: The data show that the intracellular localization of PLD1 is consistent with a role in vesicle trafficking from the Golgi apparatus and suggest that it also functions in the cell nucleus.
Abstract: Phospholipase D (PLD) hydrolyzes phosphatidylcholine to generate phosphatidic acid. In mammalian cells this reaction has been implicated in the recruitment of coatomer to Golgi membranes and release of nascent secretory vesicles from the trans-Golgi network. These observations suggest that PLD is associated with the Golgi complex; however, to date, because of its low abundance, the intracellular localization of PLD has been characterized only indirectly through overexpression of chimeric proteins. We have used highly sensitive antibodies to PLD1 together with immunofluorescence and immunogold electron microscopy as well as cell fractionation to identify the intracellular localization of endogenous PLD1 in several cell types. Although PLD1 had a diffuse staining pattern, it was enriched significantly in the Golgi apparatus and was also present in cell nuclei. On fragmentation of the Golgi apparatus by treatment with nocodazole, PLD1 closely associated with membrane fragments, whereas after inhibition of PA synthesis, PLD1 dissociated from the membranes. Overexpression of an hemagglutinin-tagged form of PLD1 resulted in displacement of the endogenous enzyme from its perinuclear localization to large vesicular structures. Surprisingly, when the Golgi apparatus collapsed in response to brefeldin A, the nuclear localization of PLD1 was enhanced significantly. Our data show that the intracellular localization of PLD1 is consistent with a role in vesicle trafficking from the Golgi apparatus and suggest that it also functions in the cell nucleus.
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TL;DR: This review focuses on the lipid precursors and products of mammalian PLD metabolism, especially phosphatidic acid and the roles this lipid performs in the mediation of the functions of PLD.
Abstract: Phospholipase D (PLD) hydrolyzes the phosphodiester bond of the glycerolipid phosphatidylcholine, resulting in the production of phosphatidic acid and free choline. Phosphatidic acid is widely considered to be the intracellular lipid mediator of many of the biological functions attributed to PLD. However, phosphatidic acid is a tightly regulated lipid in cells and can be converted to other potentially bioactive lipids, including diacylglycerol and lysophosphatidic acid. PLD activities have been described in multiple organisms, including plants, mammals, bacteria and yeast. In mammalian systems, PLD activity regulates the actin cytoskeleton, vesicle trafficking for secretion and endocytosis, and receptor signaling. PLD is in turn regulated by phosphatidylinositol-4,5-bisphosphate, protein kinase C and ADP Ribosylation Factor and Rho family GTPases. This review focuses on the lipid precursors and products of mammalian PLD metabolism, especially phosphatidic acid and the roles this lipid performs in the mediation of the functions of PLD.
458 citations
TL;DR: It is demonstrated that a regulatory pathway for mTOR signaling that involves phospholipase D (PLD) and the lipid second messenger phosphatidic acid (PA) plays a critical role in the mechanical activation of m TOR signaling, and it is shown thatmTOR signaling was partially resistant to rapamycin in muscles subjected to mechanical stimulation.
Abstract: Signaling by the mammalian target of rapamycin (mTOR) has been reported to be necessary for mechanical load-induced growth of skeletal muscle. The mechanisms involved in the mechanical activation of mTOR signaling are not known, but several studies indicate that a unique [phosphotidylinositol-3-kinase (PI3K)- and nutrient-independent] mechanism is involved. In this study, we have demonstrated that a regulatory pathway for mTOR signaling that involves phospholipase D (PLD) and the lipid second messenger phosphatidic acid (PA) plays a critical role in the mechanical activation of mTOR signaling. First, an elevation in PA concentration was sufficient for the activation of mTOR signaling. Second, the isozymes of PLD (PLD1 and PLD2) are localized to the z-band in skeletal muscle (a critical site of mechanical force transmission). Third, mechanical stimulation of skeletal muscle with intermittent passive stretch ex vivo induced PLD activation, PA accumulation, and mTOR signaling. Finally, pharmacological inhibition of PLD blocked the mechanically induced increase in PA and the activation of mTOR signaling. Combined, these results indicate that mechanical stimuli activate mTOR signaling through a PLD-dependent increase in PA. Furthermore, we showed that mTOR signaling was partially resistant to rapamycin in muscles subjected to mechanical stimulation. Because rapamycin and PA compete for binding to the FRB domain on mTOR, these results suggest that mechanical stimuli activate mTOR signaling through an enhanced binding of PA to the FRB domain on mTOR.
308 citations
TL;DR: The assembly and fusion of lipid droplets, and the processes involved in the secretion of triglycerides are reviewed, to review the importance of lipid Droplets in the development of insulin resistance and atherosclerosis.
252 citations
Cites background from "Intracellular Localization of Phosp..."
...membranes [68,69] while PLD2 is confined to the plasma membrane [68,70]....
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230 citations
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References
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TL;DR: It is suggested that BFA disrupts a dynamic membrane-recycling pathway between the ER and cis/medial Golgi, effectively blocking membrane transport out of but not back to the ER.
1,655 citations
"Intracellular Localization of Phosp..." refers result in this paper
...Instead, they showed a very diffuse localization consistent with an ER localization, confirming previous observations (Lippincott-Schwartz et al., 1989)....
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TL;DR: Growing evidence suggests that phosphorylation-dephosphorylation of the polar heads of phosphoinositides in specific intracellular locations signals either the recruitment or the activation of proteins essential for vesicular transport.
Abstract: Phosphorylated products of phosphatidylinositol play critical roles in the regulation of membrane traffic, in addition to their classical roles as second messengers in signal transduction at the cell surface. Growing evidence suggests that phosphorylation-dephosphorylation of the polar heads of phosphoinositides (polyphosphorylated inositol lipids) in specific intracellular locations signals either the recruitment or the activation of proteins essential for vesicular transport. Cross talk between phosphatidylinositol metabolites and guanosine triphosphatases is an important feature of these regulatory mechanisms.
724 citations
"Intracellular Localization of Phosp..." refers background in this paper
...Inositol phospholipids play key roles not only in mediating signal transduction events but also in regulating intracellular vesicular transport (De Camilli et al., 1996)....
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TL;DR: PLD2 is a newly identified mammalian PLD isoform with novel regulatory properties that provokes cortical reorganization and undergoes redistribution in serum-stimulated cells, suggesting that it may have a role in signal-induced cytoskeletal regulation and/or endocytosis.
691 citations
"Intracellular Localization of Phosp..." refers background in this paper
...To test this idea, an expression plasmid encoding HA-tagged PLD1 (Colley et al., 1997) was transiently transfected into NRK cells, and the localization of total PLD1 was compared with that of the exogenously expressed enzyme (Figure 8)....
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...Our data confirmed these findings when HA-tagged PLD1 was overexpressed in NRK cells and demonstrated that the distribution of the enzyme was significantly different from endogenous PLD1 (Figure 8)....
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...Two major isoforms of mammalian PLD—PLD1 and PLD2—have been characterized (Hammond et al., 1995; Colley et al., 1997)....
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...PLD2, which is regulated by different signaling molecules from PLD1, is associated with the plasma membrane (Colley et al., 1997)....
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...To test this idea, an expression plasmid encoding HA-tagged PLD1 (Colley et al., 1997) was transiently transfected into NRK cells, and the localization of total PLD1 was compared with that of the exogenously expressed enzyme (Figure 8)....
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TL;DR: The identification of the first human PLD cDNA is reported, which defines a new and highly conserved gene family and likely encodes the gene product responsible for the most widely studied endogenous PLD activity.
642 citations
"Intracellular Localization of Phosp..." refers background in this paper
...Two major isoforms of mammalian PLD—PLD1 and PLD2—have been characterized (Hammond et al., 1995; Colley et al., 1997)....
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TL;DR: An evolutionarily conserved shorter splice variant of PLD1 lacking 38 amino acids is identified that arises from regulated splicing of an alternate exon, suggesting that these three classes of regulators interact with different sites on the enzyme.
520 citations
"Intracellular Localization of Phosp..." refers background in this paper
...To date, three forms of human PLD have been characterized: PLD1a is a cytosolic enzyme that is tightly bound to membranes, and a shorter splice variant, PLD1b, has similar properties (Hammond et al., 1997)....
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