Methotrexate inhibits proteolysis of dihydrofolate reductase by the N-end rule pathway

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. In eukaryotes, the N-end rule pathway is a ubiquitin-dependent, proteasome-based system that targets and processively degrades proteins bearing certain N-terminal residues. Arg-DHFR, a modified dihydrofolate reductase bearing an N-terminal arginine (destabilizing residue in the N-end rule), is short lived in ATP-supplemented reticulocyte extract. It is shown here that methotrexate, which is a folic acid analog and high affinity ligand of DHFR, inhibits the degradation but not ubiquitination of Arg-DHFR by the N-end rule pathway. The degradation of other N-end rule substrates is not affected by methotrexate. We discuss implications of these results for the mechanism of proteasome-mediated protein degradation.