Inhibition of endosome function in CHO cells bearing a temperature-sensitive defect in the coatomer (COPI) component ε-COP
Inhibition of endosome function in CHO cells bearing a temperature-sensitive defect in the coatomer (COPI) component ε-COP
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DOI：
10.1083/jcb.139.7.1747
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发表时间：
1997-12-29
影响因子：
7.8
通讯作者：
Mellman, I
中科院分区：
文献类型：
--
作者：
Daro, E;Sheff, D;Mellman, I
最近的证据表明，在哺乳动物细胞中，外套蛋白（COPI）复合物的亚基在功能上与内体相关。我们现在提供的遗传证据表明，COPI在完整细胞的内吞作用中发挥作用。ldlF突变CHO细胞系在COPI亚基ε-COP中具有温度敏感性缺陷。除了在分泌途径中表现出条件性缺陷之外，我们发现细胞在介导内体相关功能方面也有缺陷。如对于用抗COPI抗体显微注射的细胞所发现的，限制性温度下的ldlF细胞不能被需要递送至酸性内体以渗透到胞质溶胶中的水泡性口炎（VSV）或塞姆利基森林病毒（SFV）感染。尽管受体结合转铁蛋白（Tfn）的内化没有温度敏感性缺陷，但在限制性温度下，Tfn的回收和HRP的积累受到显着抑制。分选受体结合的标志物，如EGF的溶酶体也减少了，虽然交付的流体相标志物仅部分抑制。此外，溶酶体从其典型的核周位置重新分布到ldlF细胞的尖端。突变表型开始出现在2小时内的温度变化，所需的时间为可检测的ε-COP的损失，这表明内吞缺陷不是继发于分泌途径中的一个块。重要的是，突变体表型也通过转染野生型epsilon-COP，cDNA来校正，证明它们直接或间接反映了epsilon-COP缺陷。总之，结果表明，epsilon-COP在内吞途径的早期起作用，最有可能抑制早期内体的正常分选和再循环功能。
Recent evidence has suggested that subunits of the coatomer protein (COPI) complexes are functionally associated with endosomes in mammalian cells. We now provide genetic evidence that COPI plays a role in endocytosis in intact cells. The ldlF mutant CHO cell line bears a temperature-sensitive defect in the COPI subunit epsilon-COP. In addition to exhibiting conditional defects in the secretory pathway, we find that the cells are also defective at mediating endosome-associated functions, As found for cells microinjected with anti-COPI antibodies, ldlF cells at the restrictive temperature could not be infected by vesicular stomatitis (VSV) or Semliki Forest virus (SFV) that require delivery to acidic endosomes to penetrate into the cytosol. Although there was no temperature-sensitive defect in the internalization of receptor-bound transferrin (Tfn), Tfn recycling and accumulation of HRP were markedly inhibited at the restrictive temperature. Sorting of receptor-bound markers such as EGF to lysosomes was also reduced, although delivery of fluid-phase markers was only partially inhibited. In addition, lysosomes redistributed from their typical perinuclear location to the tips of the ldlF cells. Mutant phenotypes began to emerge within 2 h of temperature shift, the time required for the loss of detectable epsilon-COP, suggesting that the endocytic defects were not secondary to a block in the secretory pathway. Importantly, the mutant phenotypes were also corrected by transfection of wild-type epsilon-COP, cDNA demonstrating that they directly or indirectly reflected the epsilon-COP defect. Taken together, the results suggest that epsilon-COP acts early in the endocytic pathway, most likely inhibiting the normal sorting and recycling functions of early endosomes.