Abstract
Phospholipase D (PLD) hydrolyses phosphatidylcholine to produce phosphatidic acid (PA) and choline. It has two isoforms, PLD1 and PLD2, which are differentially expressed depending on the cell type. In mast cells it plays an important role in signal transduction. The aim of the present study was to clarify the role of PLD2 in the secretory pathway. RBL-2H3 cells, a mast cell line, transfected to overexpress catalytically active (PLD2CA) and inactive (PLD2CI) forms of PLD2 were used. Previous observations showed that the Golgi complex was well organized in CA cells, but was disorganized and dispersed in CI cells. Furthermore, in CI cells, the microtubule organizing center was difficult to identify and the microtubules were disorganized. These previous observations demonstrated that PLD2 is important for maintaining the morphology and organization of the Golgi complex. To further understand the role of PLD2 in secretory and vesicular trafficking, the role of PLD2 in the secretory process was investigated. Incorporation of sialic acid was used to follow the synthesis and transport of glycoconjugates in the cell lines. The modified sialic acid was subsequently detected by labeling with a fluorophore or biotin to visualize the localization of the molecule after a pulse-chase for various times. Glycoconjugate trafficking was slower in the CI cells and labeled glycans took longer to reach the plasma membrane. Furthermore, in CI cells sialic acid glycans remained at the plasma membrane for longer periods of time compared to RBL-2H3 cells. These results suggest that PLD2 activity plays an important role in regulating glycoconjugate trafficking in mast cells.
Highlights
Phospholipase D (PLD) has been implicated in different cellular functions that can be attributed either to its catalytic activity or direct interaction with other proteins [1, 2]
ManNAz is incorporated into glycoproteins in the biosynthetic secretory pathway In order to determine if PLD2 plays a role in glycoprotein synthesis, the biosynthetic secretory pathway through the Golgi complex was investigated
In order to confirm if the modified sugars were being incorporated into cell glycoproteins, PLD2CA, PLD2CI and RBL-2H3 cells were incubated in presence of ManNAz or GlcNAz and glycoproteins were analyzed from whole cell lysates (Fig 1A–1C and S1A and S1B Fig)
Summary
PLD has been implicated in different cellular functions that can be attributed either to its catalytic activity or direct interaction with other proteins [1, 2]. PLD’s enzymatic activity hydrolyzes phosphatidylcholine that results in phosphatidic acid. In mammals there are two isoforms, PLD1 and PLD2 which have a 50% homology, but play distinct roles depending on the cell type [3,4,5,6,7,8]. Blockage of PLD activity with a primary alcohol results in the arrest of vesicle transport from the ER to the Golgi complex, vesicle formation at the TGN PLD2 Participates in Mast Cell Vesicular Trafficking
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