Acharya U, McCaffery J M, Jacobs R, Malhotra V
Department of Biology, University of California, San Diego, La Jolla 92093-0347, USA.
J Cell Biol. 1995 May;129(3):577-89. doi: 10.1083/jcb.129.3.577.
We have developed an in vitro system to study the biochemical events in the fusion of ilimaquinone (IQ) induced vesiculated Golgi membranes (VGMs) into stacks of cisternae. The Golgi complex in intact normal rat kidney cells (NRK) is vesiculated by treatment with IQ. The cells are washed to remove the drug and then permeabilized by a rapid freeze-thaw procedure. VGMs of 60 nm average diameter assemble into stacks of Golgi cisternae by a process that is temperature dependent, requires ATP and a high speed supernatant from cell extract (cytosol), as revealed by immunofluorescence and electron microscopy. The newly assembled stacks are functionally active in vesicular protein transport and contain processing enzymes that carry out Golgi specific modifications of glycoproteins. The fusion of VGMs requires NSF, a protein known to promote fusion of transport vesicles with the target membrane in the exocytic and endocytic pathways. Immunoelectron microscopy using Golgi specific anti-mannosidase II antibody reveals that VGMs undergo sequential changes in their morphology, whereby they first fuse to form larger vesicles of 200-300-nm average diameter which subsequently extend into tubular elements and finally assemble into stacks of cisternae.
我们开发了一种体外系统，用于研究伊利马醌（IQ）诱导的囊泡化高尔基体膜（VGMs）融合形成扁平囊堆叠过程中的生化事件。完整正常大鼠肾细胞（NRK）中的高尔基体复合物通过IQ处理而囊泡化。细胞经洗涤以去除药物，然后通过快速冻融程序使其通透化。平均直径为60 nm的VGMs通过一个温度依赖性过程组装成高尔基体扁平囊堆叠，该过程需要ATP和细胞提取物（胞质溶胶）的高速上清液，免疫荧光和电子显微镜观察揭示了这一点。新组装的堆叠在囊泡蛋白运输方面具有功能活性，并含有对糖蛋白进行高尔基体特异性修饰的加工酶。VGMs的融合需要NSF，这是一种已知能促进运输囊泡与胞吐和胞吞途径中的靶膜融合的蛋白质。使用高尔基体特异性抗甘露糖苷酶II抗体的免疫电子显微镜观察显示，VGMs的形态发生了一系列变化，即它们首先融合形成平均直径为200 - 300 nm的较大囊泡，这些囊泡随后延伸成管状结构，最终组装成扁平囊堆叠。
