Ling W L, Siddhanta A, Shields D
Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York, 10461, USA.
Methods. 1998 Oct;16(2):141-9. doi: 10.1006/meth.1998.0661.
To investigate the mechanism of secretory granule biogenesis in endocrine cells, our laboratory used rat anterior pituitary GH3 cells which secrete growth hormone and prolactin. Here we describe a simple and rapid procedure for generating permeabilized cells to dissect molecular mechanisms involved in nascent secretory vesicle budding from the trans-Golgi network (TGN). Using this system, we demonstrate that vesicle budding is temperature, energy, and cytosol dependent; in addition, cytosol from a variety of cells, including yeast (Saccharomyces cerevisiae), can support vesicle release. The budding of nascent secretory vesicles from the TGN is stimulated by a phospholipase D activity that is associated with Golgi membranes. Our results suggest that phospholipid metabolism plays an important role in the release of nascent secretory vesicles from the TGN.
为了研究内分泌细胞中分泌颗粒生物发生的机制，我们实验室使用了分泌生长激素和催乳素的大鼠垂体前叶GH3细胞。在此，我们描述了一种简单快速的方法来制备通透细胞，以剖析从反式高尔基体网络（TGN）新生分泌囊泡出芽所涉及的分子机制。利用该系统，我们证明囊泡出芽依赖于温度、能量和胞质溶胶；此外，包括酵母（酿酒酵母）在内的多种细胞的胞质溶胶都能支持囊泡释放。TGN新生分泌囊泡的出芽受到与高尔基体膜相关的磷脂酶D活性的刺激。我们的结果表明，磷脂代谢在TGN新生分泌囊泡的释放中起重要作用。
