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.

Phospholipase D: a lipid centric review.

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.

https://www.pnas.org/content/pnas/103/12/4741.full.pdf

The role of phospholipase D and phosphatidic acid in the mechanical activation of mTOR signaling in skeletal muscle.

Proteins that make, consume, and bind to phosphoinositides are important for constitutive membrane traffic. Different phosphoinositides are concentrated in different parts of the central vacuolar pathway, with phosphatidylinositol 4-phosphate predominate on Golgi, phosphatidylinositol 4,5-bisphosphate predominate at the plasma membrane, phosphatidylinositol 3-phosphate the major phosphoinositide on early endosomes, and phosphatidylinositol 3,5-bisphosphate found on late endocytic organelles. This spatial segregation may be the mechanism by which the direction of membrane traffic is controlled. Phosphoinositides increase the affinity of membranes for peripheral membrane proteins that function for sorting protein cargo or for the docking and fusion of transport vesicles. This implies that constitutive membrane traffic may be regulated by the mechanisms that control the activity of the enzymes that produce and consume phosphoinositides. Although the lipid kinases and phosphatases that function in constitutive membrane traffic are beginning to be identified, their regulation is poorly understood.

https://www.physiology.org/doi/pdf/10.1152/physrev.00033.2003

Phosphoinositides in constitutive membrane traffic

Lipid droplets as dynamic organelles connecting storage and efflux of lipids.

Phospholipase D-structure, regulation and function.

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.

Intracellular Localization of Phospholipase D1 in Mammalian Cells

Fragmentation and re-assembly of the Golgi apparatus in vitro. A requirement for phosphatidic acid and phosphatidylinositol 4,5-bisphosphate synthesis.

David A. Sweeney

Papers

Intracellular Localization of Phospholipase D1 in Mammalian Cells

Fragmentation and re-assembly of the Golgi apparatus in vitro. A requirement for phosphatidic acid and phosphatidylinositol 4,5-bisphosphate synthesis.