Use of a reporter transgene to generate arabidopsis mutants in ubiquitin-dependent protein degradation.

Ubiquitin-dependent proteolysis is a major proteolytic pathway in the cytoplasm and nucleus of eukaryotic cells. We introduced a gene encoding a substrate for this pathway into the genome of Arabidopsis thaliana. The transgene codes for a hybrid protein consisting of dihydrofolate reductase (DHFR, EC 1.5.1.3) fused to a degradation signal that is specifically recognized by components of the ubiquitin-dependent proteolysis pathway. Elevated concentrations of the DHFR protein confer resistance to the drug methotrexate, but rapid degradation prevents accumulation of the protein in the plant. Therefore, transgenic A. thaliana lines expressing the DHFR fusion protein are methotrexate-sensitive. Selection for mutants resistant to methotrexate resulted in plants impaired in degradation of the DHFR model substrate, as shown by an increase in protein level in the mutants.