Characterization of phospholipid synthesis in maintaining the structural integrity of the Golgi apparatus
Sweeney, David A.
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Recent work from our laboratory demonstrated that phosphatidic acid (PA) and phosphatidylinositol 4,5-bisphosphate (Ptdlns(4,5)P2), are required to maintain the structural integrity of the Golgi apparatus (GA). To investigate the role of these lipids in regulating Golgi structure and function, we developed a novel assay to follow the release of post-Golgi vesicles. Isolated rat liver Golgi membranes were incubated with 3H-CMP-sialic acid to radiolabel and follow the exit of endogenous soluble and membrane glycoproteins present in the late Golgi and trans-Golgi network at the time of purification. The release of post-Golgi secretory vesicles was determined by measuring the incorporation of 3H-labeled proteins into a medium speed supernatant. Vesicle budding was temperature, cytosol energy and time dependent. These labeled N-linked glycoproteins were contained in membrane bound vesicles as indicated by their resistance to protease digestion and sedimentation profile. The release kinetics of a number of resident glycoproteins were examined. The exit profile for TGN 38, a resident Golgi protein, was revealed to be slower than either the average release of total glycoprotein or the very robust release of the endosomal system protein transferrin. Electron microscopy of Golgi fractions prior to and after incubation under vesicle budding conditions demonstrated that the stacked Golgi cisternae generated a heterogeneous population of vesicles (50--350 nm diameter). These data indicate the strong selectivity thought to reside in the late Golgi. Vesicle production was stimulated in the presence of exogenous phospholipase D indicating the involvement of phosphatidic acid. Inhibition of phospholipase D-mediated PA synthesis via the transphosphatidylation properties of PLD by 1-butanol, resulted in the >95% fragmentation of the Golgi membranes in vitro into 50--100 nm vesicles as assessed by morphometry; this correlated with diminished Ptdlns(4,5)P2 synthesis. Following alcohol washout, PA synthesis resumed and in the presence of cytosol and ATP Ptdlns(4,5)P 2 synthesis was restored. Under these conditions approximately 30% of the fragmented Golgi elements reformed into flattened cisternae and strikingly when the re-assembled Golgi membranes were incubated under vesicle budding conditions release of nascent vesicles was restored. It has been known for 30 years that the GA fragmented at the onset of mitosis only to reform again in daughter cells. Significantly, fragmentation of the Golgi during mitosis also correlated with inhibition of Ptdlns(4,5)P2 synthesis. The reformation of those mitotically fragmented membranes also required the concomitant return of Ptdlns(4,5)P2 synthesis to reform. These data demonstrate that inositol phospholipid synthesis is essential for the structure and function of the Golgi apparatus.