Intracellular localization of phospholipase D1 in mammalian cells

Phospholipase D (PLD) hydrolyzes phosphatidylcholine to generate phosphatid ic acid. In mammalian cells this reaction has been implicated in the recrui tment of coatomer to Golgi membranes and release of nascent secretory vesic les from the trans-Golgi network. These observations suggest that PLD is as sociated with the Golgi complex; however, to date, because of its low abund ance, the intracellular localization of PLD has been characterized only ind irectly through overexpression of chimeric proteins. We have used highly se nsitive antibodies to PLD1 together with immunofluorescence and immunogold electron microscopy as well as cell fractionation to identify the intracell ular localization of endogenous PLD1 in several cell types. Although PLD1 h ad a diffuse staining pattern, it was enriched significantly in the Golgi a pparatus and was also present in cell nuclei. On fragmentation of the Golgi apparatus by treatment with nocodazole, PLD1 closely associated with membr ane fragments, whereas after inhibition of PA synthesis, PLD1 dissociated f rom the membranes. Overexpression of an hemagglutinin-tagged form of PLD1 r esulted in displacement of the endogenous enzyme from its perinuclear local ization to large vesicular structures. Surprisingly, when the Golgi apparat us collapsed in response to brefeldin A, the nuclear localization of PLD1 w as enhanced significantly. Our data show that the intracellular localizatio n of PLD1 is consistent with a role in vesicle trafficking from the Golgi a pparatus and suggest that it also functions in the cell nucleus.