Degradation of a nuclear-localized protein in mammalian COS cells, using Escherichia coli beta-galactosidase as a model protein.
To investigate the mechanism of degradation of proteins localized in the nucleus, we constructed genes encoding modified Escherichia coli beta-galactosidases and expressed them in mammalian COS cells. When the beta-galactosidase with a nuclear localization signal from SV 40 T antigen was expressed in COS cells, the beta-galactosidase polypeptide was localized in the nuclei and was stable for at least 4 h. When 16 amino acid residues were deleted from the C-terminal end, the beta-galactosidase polypeptide was also observed in the nuclei but it was degraded rapidly, with a half-life of 1.6 h. When the nuclear localizing signal was replaced with a mutant sequence, which lacks nuclear targeting activity, the beta-galactosidase polypeptides were present throughout the cells rather than in the nuclei. The beta-galactosidase polypeptide with the complete C terminus was stable and the cytoplasmic truncated polypeptide was degraded at the same rate as the nuclear C terminus truncated polypeptide. The beta-galactosidase polypeptides with the complete C terminus were present as a tetramer as reported previously and had beta-galactosidase activity, but the C terminus truncated polypeptides were present as monomer and had no enzyme activity, indicating that C terminus truncated beta-galactosidase is malfolded. Together, the results suggest that a nuclear-localized malfolded protein is degraded as rapidly as a cytoplasmic malfolded protein.
Tsuneoka
M
M
Institute of Life Science, Kurume University, Fukuoka, Japan.
Mekada
E
E
D000595:Amino Acid Sequence; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D016521:Electrophoresis, Gel, Pulsed-Field; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005455:Fluorescent Antibody Technique; D008322:Mammals; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D011487:Protein Conformation; D016271:Proto-Oncogene Proteins c-myc / Q000032:analysis; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
D000952:Antigens, Polyomavirus Transforming; D009687:Nuclear Proteins; D016271:Proto-Oncogene Proteins c-myc; D001616:beta-Galactosidase