期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 277, 期 4, 页码 3030-3039出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M104639200
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资金
- NCI NIH HHS [P30CA13330] Funding Source: Medline
- NIDDK NIH HHS [DK21860, DK P30 41296, DK07513] Funding Source: Medline
Recent work from our laboratory demonstrated that phosphatidic acid (PA) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P-2), are required to maintain the structural integrity of the Golgi apparatus. 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 [H-3]CMP sialic acid to radiolabel endogenous soluble and membrane glycoproteins present in the late Golgi and trans-Golgi network. The release of post-Golgi secretory vesicles was determined by measuring incorporation of H-3-labeled proteins into a medium speed supernatant. Vesicle budding was dependent on temperature, cytosol, energy and time. Electron microscopy of Golgi fractions prior to and after incubation demonstrated that the stacked Golgi cisternae generated a heterogeneous population of vesicles (50- to 350-nm diameter). Inhibition of phospholipase D-mediated PA synthesis, by incubation with 1-butanol, resulted in the complete fragmentation of the Golgi membranes in vitro into 50- to 100-nm vesicles; this correlated with diminished PtdIns(4,5)P-2 synthesis. Following alcohol washout, PA synthesis resumed and in the presence of cytosol PtdIns(4,5)P-2 synthesis was restored. Most significantly, under these conditions the fragmented Golgi elements reformed into flattened cisternae and the re-assembled Golgi supported vesicle release. These data demonstrate that inositol phospholipid synthesis is essential for the structure and function of the Golgi apparatus.
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