Intracellular Localization of Phospholipase D1 in Mammalian Cells

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.

Phospholipases D1 and D2 Coordinately Regulate Macrophage Phagocytosis

“…Western blotting of the IPs confirmed the specificity of the Abs, (Fig. 2B), as previously reported (28,49,50). No PLD1 protein was detected in the anti-PLD2 IPs, and only a minor amount of PLD2 was detected in the anti-PLD1 IPs.…”

“…The cortical distribution is consistent with the plasma membrane localization proposed from biochemical studies of phagocytes (16, 32, 48, 56 -58). The intracellular PLD1 is likely vesicular in origin, in agreement with data from several cell types (28,49,58). PLD2 localized to intracellular vesicles (especially in the perinuclear region) and the nucleus in resting macrophages (Fig.…”

“…In bovine adrenal chromaffin cells and rat cerebellar granule cells, PLD1 is localized predominantly to the plasma membrane (28,62), whereas in rat liver homogenates PLD1 is detected in the both the Golgi apparatus and plasma membrane upon subcellular fractionation (49). In cell lines, PLD1 has been localized to intracellular membranes (Golgi, endosomes, lysosomes, and nucleus) as well as the plasma membrane (28,49,57,63). Transfection of chromophore-or epitope-tagged PLD1 has demonstrated a similar range of subcellular distributions, along with evidence of activation-associated translocation of PLD1 from secretory vesicles to the plasma membrane (58, 64 -66).…”

“…Transfection of chromophore-or epitope-tagged PLD1 has demonstrated a similar range of subcellular distributions, along with evidence of activation-associated translocation of PLD1 from secretory vesicles to the plasma membrane (58, 64 -66). Conflicting evidence exists concerning whether overexpression of PLD proteins may (49,50,66) be accompanied by aberrant subcellular localization.…”

mTOR signaling: PLD takes center stage

“…32,35 This is not only in keeping with the known regulation of PLD1 by mitogens, but also consistent with the subcellular localization of PLD1 to perinuclear regions where both Rheb and mTOR are found. 66,69,70 Is PLD2 also involved in mTOR signaling? The reported plasma membrane localization of PLD2, 69,71 seems to disagree with such a role.…”