Robbins A R, Oliver C, Bateman J L, Krag S S, Galloway C J, Mellman I
J Cell Biol. 1984 Oct;99(4 Pt 1):1296-308. doi: 10.1083/jcb.99.4.1296.
A Chinese hamster ovary cell mutant DTG 1-5-4, was selected for pleiotropic defects in receptor-mediated endocytosis by methods previously described (Robbins, A. R., S. S. Peng, and J. L. Marshall, 1983, J. Cell Biol., 96:1064-1071). DTG 1-5-4 exhibited increased resistance to modeccin, Pseudomonas toxin, diphtheria toxin, Sindbis virus, and vesicular stomatitis virus, as well as decreased uptake via the mannose 6-phosphate receptor. Fluorescein-dextran-labeled endosomes isolated from DTG 1-5-4 were deficient in ATP-dependent acidification in vitro. Endocytosis and endosome acidification were both restored in revertants of DTG 1-5-4 and in hybrids of DTG 1-5-4 with DTF 1-5-1, another endocytosis mutant exhibiting decreased ATP-dependent endosome acidification. Both DTG 1-5-4 and DTF 1-5-1 were blocked at two stages of infection with Sindbis virus: at low multiplicities of infecting virus, resistance reflected a block in viral penetration into the cytoplasm, but at higher multiplicities of infection the block was in virus release. Like endocytosis, release of Sindbis virus was increased in revertants of DTG 1-5-4 and in DTG 1-5-4 X DTF 1-5-1 hybrids. Decreased release of virus from DTG 1-5-4 correlated with defects in some of the Golgi apparatus-associated steps of Sindbis glycoprotein maturation: proteolytic processing of the precursor pE2, galactosylation, and transport to the cell surface all were inhibited. In contrast, mannosylation, fucosylation, and acylation of the Sindbis glycoproteins, and galactosylation of vesicular stomatitis virus and cellular glycoproteins occurred to similar respective extents in mutant and parent. Electron microscopic examination of Sindbis-infected DTG 1-5-4 showed a remarkable accumulation of nucleocapsids bound to cisternae adjacent to the Golgi apparatus; virions were observed in the lumina of some of these cisternae. That the alterations in both endocytosis and Golgi-associated steps of viral maturation result from a single genetic lesion indicates that these processes are dependent on a common biochemical mechanism. We suggest that endocytic and secretory pathways may share a common component involved in ion transport.
通过先前描述的方法（Robbins, A. R., S. S. Peng, and J. L. Marshall, 1983, J. Cell Biol., 96:1064 - 1071），选择了一株中国仓鼠卵巢细胞突变体DTG 1 - 5 - 4，其在受体介导的内吞作用中存在多效性缺陷。DTG 1 - 5 - 4对相思豆毒素、铜绿假单胞菌毒素、白喉毒素、辛德毕斯病毒和水疱性口炎病毒的抗性增强，同时通过甘露糖6 - 磷酸受体的摄取减少。从DTG 1 - 5 - 4分离的荧光素 - 葡聚糖标记的内体在体外缺乏ATP依赖性酸化。在DTG 1 - 5 - 4的回复突变体以及DTG 1 - 5 - 4与DTF 1 - 5 - 1（另一个内吞作用突变体，其ATP依赖性内体酸化降低）的杂交体中，内吞作用和内体酸化均得以恢复。DTG 1 - 5 - 4和DTF 1 - 5 - 1在辛德毕斯病毒感染的两个阶段均受到阻断：在低感染复数时，抗性反映了病毒进入细胞质的阻断，但在高感染复数时，阻断发生在病毒释放阶段。与内吞作用一样，DTG 1 - 5 - 4的回复突变体以及DTG 1 - 5 - 4与DTF 1 - 5 - 1的杂交体中辛德毕斯病毒的释放增加。DTG 1 - 5 - 4中病毒释放减少与辛德毕斯糖蛋白成熟过程中一些与高尔基体相关步骤的缺陷相关：前体pE2的蛋白水解加工、半乳糖基化以及向细胞表面的转运均受到抑制。相比之下，辛德毕斯糖蛋白的甘露糖基化、岩藻糖基化和酰化，以及水疱性口炎病毒和细胞糖蛋白的半乳糖基化在突变体和亲本中的程度相似。对感染辛德毕斯病毒的DTG 1 - 5 - 4进行电子显微镜检查发现，与高尔基体相邻的扁平囊泡上显著积累了与核衣壳结合的物质；在其中一些扁平囊泡的腔内观察到了病毒粒子。内吞作用和病毒成熟的高尔基体相关步骤的改变是由单一遗传损伤引起的，这表明这些过程依赖于共同的生化机制。我们认为内吞和分泌途径可能共享一个参与离子转运的共同成分。
