Guo Q, Vasile E, Krieger M
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
J Cell Biol. 1994 Jun;125(6):1213-24. doi: 10.1083/jcb.125.6.1213.
The CHO cell temperature-sensitive mutant ldlF exhibits two defects in membrane traffic at the nonpermissive temperature (39.5 degrees C): rapid degradation of LDL receptors, possibly caused by endocytic missorting, and disruption of ER-through-Golgi transport. Here, we show that at 39.5 degrees C, the Golgi in ldlF cells dissociated into vesicles and tubules. This dissociation was inhibited by AlF4-, suggesting trimeric G proteins are involved in the dissociation mechanism. This resembled the effects of brefeldin A on wild-type cells. We isolated a hamster cDNA that specifically corrected the ts defects of ldlF cells, but not those of other similar ts mutants (ldlE, ldlG, ldlH, and End4). Its predicted protein sequence is conserved in humans, rice, Arabidopsis, and Caenorhabditis elegans, and is virtually identical to that of bovine epsilon-COP, a component of the coatomer complex implicated in membrane transport. This provides the first genetic evidence that coatomers in animal cells can play a role both in maintaining Golgi structure and in mediating ER-through-Golgi transport, and can influence normal endocytic recycling of LDL receptors. Thus, along with biochemical and yeast genetics methods, mammalian somatic cell mutants can provide powerful tools for the elucidation of the mechanisms underlying intracellular membrane traffic.
中国仓鼠卵巢（CHO）细胞温度敏感突变体ldlF在非允许温度（39.5摄氏度）下的膜运输中表现出两个缺陷：低密度脂蛋白（LDL）受体的快速降解，可能是由内吞分选错误引起的，以及内质网到高尔基体运输的中断。在这里，我们表明，在39.5摄氏度时，ldlF细胞中的高尔基体解离成囊泡和小管。AlF4-抑制了这种解离，表明三聚体G蛋白参与了解离机制。这类似于布雷菲德菌素A对野生型细胞的影响。我们分离出一个仓鼠cDNA，它特异性地纠正了ldlF细胞的温度敏感缺陷，但不能纠正其他类似温度敏感突变体（ldlE、ldlG、ldlH和End4）的缺陷。其预测的蛋白质序列在人类、水稻、拟南芥和秀丽隐杆线虫中保守，并且与牛ε-COP几乎相同，牛ε-COP是参与膜运输的外被体复合物的一个组分。这提供了第一个遗传学证据，即动物细胞中的外被体复合物在维持高尔基体结构以及介导内质网到高尔基体的运输中发挥作用，并且可以影响LDL受体的正常内吞再循环。因此，与生化和酵母遗传学方法一起，哺乳动物体细胞突变体可以为阐明细胞内膜运输的潜在机制提供强大的工具。
