Cytosolic phospholipase A2 regulates Golgi structure and modulates intracellular trafficking of membrane proteins
Gabriel J. Choukroun, … , Dennis Brown, Joseph V. Bonventre
Gabriel J. Choukroun, … , Dennis Brown, Joseph V. Bonventre
Published October 15, 2000
Citation Information: J Clin Invest. 2000;106(8):983-993. https://doi.org/10.1172/JCI8914.
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Article

Cytosolic phospholipase A2 regulates Golgi structure and modulates intracellular trafficking of membrane proteins

Abstract

The Golgi complex and the trans-Golgi network are critical cellular organelles involved in the endocytic and biosynthetic pathways of protein trafficking. Lipids have been implicated in the regulation of membrane-protein trafficking, vesicular fusion, and targeting. We have explored the role of cytosolic group IV phospholipase A2 (cPLA2) in membrane-protein trafficking in kidney epithelial cells. Adenoviral expression of cPLA2 in LLC-PK1 kidney epithelial cells prevents constitutive trafficking to the plasma membrane of an aquaporin 2-green fluorescent protein chimera, with retention of the protein in the rough endoplasmic reticulum. Plasma membrane Na+-K+-ATPase α-subunit localization is markedly reduced in cells expressing cPLA2, whereas the trafficking of a Cl–/HCO3– anion exchanger to the plasma membrane is not altered in these cells. Expression of cPLA2 results in dispersion of giantin and β-COP from their normal, condensed Golgi localization, and in marked disruption of the Golgi cisternae. cPLA2 is present in Golgi fractions from noninfected LLC-PK1 cells and rat kidney cortex. The distribution of tubulin and actin was not altered by cPLA2, indicating that the microtubule and actin cytoskeleton remain intact. Total cellular protein synthesis is unaffected by the increase in cPLA2 activity. Thus cPLA2 plays an important role in determining Golgi architecture and selective control of constitutive membrane-protein trafficking in renal epithelial cells.

Authors

Gabriel J. Choukroun, Vladimir Marshansky, Corinne E. Gustafson, Mary McKee, Roger J. Hajjar, Anthony Rosenzweig, Dennis Brown, Joseph V. Bonventre

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Figure 1

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(a and b) Assay of cPLA2 activity in LLC-PK1 cells expressing LacZ and c...
(a and b) Assay of cPLA2 activity in LLC-PK1 cells expressing LacZ and cPLA2 and in uninfected cells. (a) AQP2-GFP(CT) LLC-PK1 cells were incubated with either AdLacZ or AdcPLA2 at different moi (10, 50, 100, 200, and 400 pfu/cell). Forty-eight hours later, the cells were lysed and centrifuged at 100,000 g for 1 hour at 4°C. Each assay was performed three times in duplicate. Cell lysates of MDCK and rat mesangial cells (MC) were processed at the same time and used to compare the level of activity in the different cell lines. cPLA2 activity was significantly greater (P < 0.01) in cells infected with AdcPLA2 at 10, 50, 100, 200, and 400 pfu/cell when compared with control noninfected cells and AdLacZ infected cells. (b) cPLA2 activity was determined 1, 2, 3, 6, 10, 15, and 21 days after infection with AdLacZ or AdcPLA2 at 50 pfu/cell or in control cells (n = 3). AdcPLA2-treated cells had significantly greater (P < 0.01) cPLA2 activity at each time point when compared with AdLacZ and control cells. (c) Immunoblot of cPLA2 from control cells and cells infected with AdLacZ and AdcPLA2. Cells were infected with AdLacZ or AdcPLA2 at different moi. Forty-eight hours later, the cells were solubilized, and 25 μg of each lysate were separated by SDS-PAGE and a Western blot generated with 1:5000 diluted polyclonal anti-cPLA2 Ab.