The C terminus of mouse ornithine decarboxylase confers rapid degradation on dihydrofolate reductase. Support for the pest hypothesis.
Several years ago, we proposed that polypeptide regions rich in proline (P), glutamic acid (E), serine (S), and threonine (T) (PEST) target intracellular proteins for destruction (Rogers, S., Wells, R., and Rechsteiner, M. (1986) Science 234, 364-368). To test the PEST hypothesis, we have produced chimeric proteins in which the N or C terminus of mouse dihydrofolate reductase is extended by the PEST-containing C terminus of mouse ornithine decarboxylase. Oligonucleotides encoding the 37 C-terminal residues of mouse ornithine decarboxylase (mODC) or equivalent lengths of dissimilar amino acids were inserted at appropriate sites in a dihydrofolate reductase (DHFR) expression vector. The various fusion proteins were expressed in Escherichia coli and purified to homogeneity by enzyme affinity chromatography. All purified fusion proteins exhibited similar abilities to convert dihydrofolate to tetrahydrofolate, thereby demonstrating that the attachment of peptide extensions to either terminus did not prevent the proper folding of DHFR. Metabolic stabilities of the radioiodinated fusion proteins were assayed in rabbit reticulocyte lysate or Xenopus egg extract. Proteolysis was found to be energy-dependent with mODC-DHFR fusion proteins being degraded from 2 to almost 40-fold faster than the parental DHFR molecule or DHFR fusion proteins bearing non-PEST extensions. Deletion of most of the PEST region from the mODC extension resulted in a significantly more stable fusion protein. Rapid proteolysis of DHFR proteins containing intact mODC extensions provides support for the PEST hypothesis.
Loetscher
P
P
Department of Biochemistry, University of Utah School of Medicine, Salt Lake City 84132.
Pratt
G
G
Rechsteiner
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002678:Chimera; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D009865:Oocytes / Q000378:metabolism; D009955:Ornithine Decarboxylase / Q000235:genetics* / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012156:Reticulocytes / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014981:Xenopus
D015345:Oligonucleotide Probes; D011993:Recombinant Fusion Proteins; D013762:Tetrahydrofolate Dehydrogenase; D009955:Ornithine Decarboxylase