A single point mutation in epsilon-COP results in temperature-sensitive, lethal defects in membrane transport in a Chinese hamster ovary cell mutant

At the nonpermissive temperature of 39.5 degrees C, the Chinese hamster ovary cell conditionally lethal, temperature-sensitive (ts) mutant IdlF 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 IdlF cells of a point mutation in the epsilon-COP gene, Glu(251) to Lys(251), which prevents the corresponding cDNA from correcting the defects in transfected IdlF 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 IdlF 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 (t(1/2) > 6 h at 34 degrees C and similar to 1-2 h at 39.5 degrees C), and this accounts for the virtual absence of detectable ts-epsilon-COP protein in IdlF cells after incubation at 39.5 degrees C for > 6 h, The steady state levels in IdlF cells of another coatomer subunit, beta-COP, and the peripheral Golgi protein IdlCp 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 IdlF cells, and the stability of beta-COP is not linked directly to that of epsilon-COP. IdlF 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.