Guo Q, Penman M, Trigatti B L, Krieger M
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.
J Biol Chem. 1996 May 10;271(19):11191-6. doi: 10.1074/jbc.271.19.11191.
At the nonpermissive temperature of 39.5 degrees C, the Chinese hamster ovary cell conditionally lethal, temperature-sensitive (ts) mutant ldlF exhibits the following defects: rapid degradation of low density lipoprotein receptors, disruption of ER-through Golgi transport, and disintegration of the Golgi apparatus. All of these are corrected by transfection with an expression vector for wild-type epsilon-COP, a subunit of coatomers (Guo, Q., Vasile, E., and Krieger, M. (1994) J. Cell Biol. 125, 1213-1224). We now report the identification in ldlF cells of a point mutation in the epsilon-COP gene, Glu251 to Lys251, which prevents the corresponding cDNA from correcting the defects in transfected ldlF cells and the immunochemical analysis of the synthesis, structure, and stability of epsilon-COP. At the permissive temperature (34 degrees C), the steady state level of ts-epsilon-COP in ldlF cells was about half that of epsilon-COP in wild-type Chinese hamster ovary cells and the isoelectric point of ts-epsilon-COP was 0.14 pH units higher than that of the wild-type protein. The stability but not the biosynthesis of ts-epsilon-COP was temperature-sensitive (t1/2 > 6 h at 34 degrees C and approximately 1-2 h at 39.5 degrees C), and this accounts for the virtual absence of detectable ts-epsilon-COP protein in ldlF cells after incubation at 39.5 degrees C for > 6h. The steady state levels in ldlF cells of another coatomer subunit, beta-COP, and the peripheral Golgi protein ldlCp were not temperature-sensitive. Thus, a mutation in epsilon-COP that causes instability at 39.5 degrees C is responsible for all of the temperature-sensitive defects in ldlF cells, and the stability of beta-COP is not linked directly to that of epsilon-COP. ldlF cells should be useful for the future analysis of the structure and function of epsilon-COP, the assembly of COPs into coatomers, and the participation of coatomers in intracellular membrane transport.
在39.5℃的非允许温度下，中国仓鼠卵巢细胞条件致死性温度敏感（ts）突变体ldlF表现出以下缺陷：低密度脂蛋白受体快速降解、内质网到高尔基体运输中断以及高尔基体解体。通过用野生型ε-COP（一种外被体亚基）的表达载体转染可纠正所有这些缺陷（Guo，Q.，Vasile，E.，和Krieger，M.（1994）J. Cell Biol. 125，1213 - 1224）。我们现在报告在ldlF细胞中鉴定出ε-COP基因中的一个点突变，即Glu251突变为Lys251，该突变阻止相应的cDNA纠正转染的ldlF细胞中的缺陷，并对ε-COP的合成、结构和稳定性进行了免疫化学分析。在允许温度（34℃）下，ldlF细胞中ts-ε-COP的稳态水平约为野生型中国仓鼠卵巢细胞中ε-COP的一半，且ts-ε-COP的等电点比野生型蛋白高0.14个pH单位。ts-ε-COP的稳定性而非生物合成是温度敏感的（在34℃时t1/2＞6小时，在39.5℃时约为1 - 2小时），这解释了在39.5℃孵育＞6小时后ldlF细胞中几乎检测不到ts-ε-COP蛋白的原因。另一种外被体亚基β-COP和高尔基体周边蛋白ldlCp在ldlF细胞中的稳态水平不是温度敏感的。因此，ε-COP中导致在39.5℃不稳定的突变是ldlF细胞中所有温度敏感缺陷的原因，且β-COP的稳定性与ε-COP的稳定性没有直接关联。ldlF细胞将有助于未来对ε-COP的结构和功能、外被体蛋白组装成外被体以及外被体在细胞内膜运输中的参与情况进行分析。
