Intracellular localization of phospholipase D1 in mammalian cells.
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.
Freyberg
Z
Z
Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Sweeney
D
D
Siddhanta
A
A
Bourgoin
S
S
Frohman
M
M
Shields
D
D
D000595:Amino Acid Sequence; D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D002460:Cell Line; D015870:Gene Expression; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D007424:Intracellular Fluid / Q000201:enzymology; D008322:Mammals; D008969:Molecular Sequence Data; D015739:Nocodazole / Q000494:pharmacology; D010739:Phospholipase D / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D013347:Subcellular Fractions
D011500:Protein Synthesis Inhibitors; D020126:Brefeldin A; D010739:Phospholipase D; C106801:phospholipase D1; D015739:Nocodazole