Zuber C, Roth J, Misteli T, Nakano A, Moremen K
Department of Cell and Molecular Pathology, University of Zürich Medical School, Switzerland.
Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9818-22. doi: 10.1073/pnas.88.21.9818.
The temperature-sensitive Chinese hamster ovary (CHO) cell mutant DS28-6 has been previously shown to be pleiotropically defective in protein secretion. We have examined the mutant cells to determine the intracellular site of the block in secretion. By transmission electron microscopy a time-dependent disassembly of the Golgi apparatus was found under nonpermissive temperature, which resulted in the loss of the cisternal stack. Complete reorganization of the Golgi apparatus occurred after shift to permissive temperature. Under nonpermissive temperature, a microtubule- and energy-dependent redistribution of Golgi mannosidase II and galactosyltransferase into a pattern characteristic of the endoplasmic reticulum (ER) was observed. Inhibition of protein synthesis by cycloheximide had no influence on Golgi mannosidase II redistribution. Evidence for Golgi apparatus-associated processing of oligosaccharides in the ER was obtained by lectin-gold cytochemistry revealing the presence of the galactose (beta 1----4)N-acetylglucosamine sequence and sialic acid residues. Furthermore, 7-nitrobenz-2-oxa-1,3-diazol-4-yl-tagged ceramide, a lipidic trans-Golgi apparatus marker in CHO cells, exhibited an energy-dependent redistribution into the ER. These effects were fully reversible upon shift to permissive temperature. Thus, mutant DS28-6 cells exhibit key features of the brefeldin A phenotype, which suggests that the observed brefeldin A effects result from interference with a normally occurring cellular process.
温度敏感型中国仓鼠卵巢（CHO）细胞突变体DS28 - 6先前已被证明在蛋白质分泌方面存在多效性缺陷。我们对突变细胞进行了检测，以确定分泌过程中受阻的细胞内位点。通过透射电子显微镜观察发现，在非允许温度下，高尔基体出现了随时间的解体，导致扁平囊堆叠结构消失。转移到允许温度后，高尔基体发生了完全重组。在非允许温度下，观察到高尔基体甘露糖苷酶II和半乳糖基转移酶以微管和能量依赖的方式重新分布成内质网（ER）特有的模式。用环己酰亚胺抑制蛋白质合成对高尔基体甘露糖苷酶II的重新分布没有影响。通过凝集素 - 金细胞化学揭示了半乳糖（β1----4）N - 乙酰葡糖胺序列和唾液酸残基的存在，从而获得了在内质网中与高尔基体相关的寡糖加工的证据。此外，CHO细胞中一种脂质型反式高尔基体标记物7 - 硝基苯 - 2 - 恶唑 - 1,3 - 二氮杂环丁烷 - 4 - 基标记的神经酰胺表现出能量依赖的向内质网的重新分布。转移到允许温度后，这些效应是完全可逆的。因此，突变体DS28 - 6细胞表现出布雷菲德菌素A表型的关键特征，这表明观察到的布雷菲德菌素A效应是由对正常细胞过程的干扰引起的。
