Involvement of sequences near both amino and carboxyl termini in the rapid intracellular degradation of tyrosine aminotransferase.
The degradation of rat liver tyrosine aminotransferase has been studied after transfection of suitable expression vectors into mammalian cells in culture. A normal rapid rate of degradation (half-life about 6 h) was observed in cells under stable transfection conditions. However, the higher enzyme levels produced during transient transfections or after amplification with methotrexate caused the apparent half-life of degradation to increase substantially. Analysis of expression in Chinese hamster ovary (CHO)-DG44 cells from vectors with deletions near either end of the tyrosine aminotransferase coding sequence showed that approximately the first 40 and the last 12 amino acid residues are not required to obtain normal catalytic function. When catalytically active deletion mutants were examined for effects on tyrosine aminotransferase degradation in stably transfected CHO-DG44 cell populations, short sequences near each end of the protein were found to be necessary for rapid degradation. The required sequence near the amino terminus is located between amino acids 30 and 40 and includes the highly basic region RKKGRKAR, a potential ubiquitin attachment site. The other essential sequence (EECDK) is located at the very COOH terminus of the 454-amino acid chain and is part of an acidic domain rich in cysteines and having PEST characteristics (rich in Pro, Glu, and Thr). Ser448, a potential casein kinase II phosphorylation site, is not required for activity or rapid degradation of tyrosine aminotransferase. No correlation was observed between the intracellular degradation rates of the various mutant proteins and their heat stabilities in vitro.
Stellwagen
R H
RH
Department of Biochemistry and Molecular Biology, University of Southern California School of Medicine, Los Angeles 90033.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D004795:Enzyme Stability; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013728:Terminator Regions, Genetic; D013816:Thermodynamics; D014162:Transfection; D014444:Tyrosine Transaminase / Q000235:genetics / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014444:Tyrosine Transaminase