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 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.
Guo
Q
Q
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.
Penman
M
M
Trigatti
B L
BL
Krieger
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001692:Biological Transport; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D020756:Coatomer Protein; D006224:Cricetinae; D005804:Genes, Lethal; D018698:Glutamic Acid; D007700:Kinetics; D008239:Lysine; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology; D017354:Point Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis; D013696:Temperature; D014162:Transfection
D000906:Antibodies; D020756:Coatomer Protein; D008565:Membrane Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D018698:Glutamic Acid; D008239:Lysine