Figure 1.

F-DHFR is an N-end rule substrate that accumulates in prt1 mutant cells. Leaf material of transgenic plants was incubated with radioactive methionine in the presence (even lanes) or the absence (odd lanes) of clasto-lactacystin β-lactone (lactacystin), a specific inhibitor of the proteasome. Proteins were extracted, and DHFR was isolated by immunoprecipitation and detected by SDS/PAGE and fluorography. The level of M-DHFR (DHFR with an extension bearing an N-terminal methionine residue) is not increased by lactacystin (lanes 1 vs. 2), whereas F-DHFR increases considerably (lanes 3 vs. 4). Lanes 5 and 6 are the same as lanes 3 and 4, but transgene is expressed from the weaker nopaline synthase promoter. Lanes 7 and 8 are the same as lanes 5 and 6, but plant has a mutation in the PRT1 gene, which results in metabolic stabilization of F-DHFR. The arrowhead marks the origin of the separation gel, two dots indicate DHFR protein bands, the lower one being either a conformer or a cleavage product. Positions of molecular weight marker bands are indicated in the middle.