Abstract
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.
Highlights
Several years ago, we proposedthat polypeptide re- differ by at least 1000-fold
We report that addition of the 37 C-terminal residues of mODC to either endof dihydrofolate reductase results in an enzymatically active fusion protein that is rapidly degraded in frog egg extract andin reticulocyte lysate
DHFR can be substantially puri- resemblance to PEST sequences
Summary
Production and Purification of DHFR Fusion ProteinsDHFR has several properties that make it an attractive recipient for PEST sequences. The enzyme is small and monomeric, and anumber of DHFR structures have been solved at high resolution by x-ray diffraction (30). Since both the N and C termini extend intosolvent as an antiparallel @-sheet, they present suitable sites for the addition of peptide extensions. Little effect on DHFR stability in yeastcells, and NP2 is the Stueber andcolleagues (21) have developed a plasmid that translation product of the NP1coding sequence inserted into contains convenient restriction sites for modifying either the N or C terminus of mouse DHFR. As shown by the data, the purified fusion proteins exhibited DHFR activity roughly comparable with the enzyme lackingP or N P extensions
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