In vitro screening for substrates of the N-end rule-dependent ubiquitylation

We describe a systematic, high-throughput approach to the discovery of protein substrates of ubiquitylation. This method uses a library of cDNAs in combination with a reticulocyte lysate-based, transcription-translation system that acts as both an excellent means for high-throughput protein expression and a source of ubiquitylation enzymes. Ubiquitylation of newly expressed proteins occurs in this milieu from the action of any one of a number of E3 ligases that are present in the lysate. Specific detection of ubiquitylated proteins is carried out using electrochemiluminescence-based assays in conjunction with a multiplexing scheme that provides replicate measurements of the ubiquitylated products and two controls in each well of a microtiter plate. We used this approach to identify putative substrates of the N-end rule-dependent ubiquitylation (mediated by the UBR family of ubiquitin ligases), a system already well known to have high endogenous activity in reticulocyte lysates. We screened a library of approximately 18,000 cDNA clones, one clone per well, by expressing them in reticulocyte lysate and measuring the extent of modification. We selected approximately 500 proteins that showed significant ubiquitylation. This set of modified proteins was redacted to approximately 60 potential substrates of the N-end rule pathway in a secondary screen that involved looking for inhibition of ubiquitylation in reticulocyte lysates supplemented with specific inhibitors of the N-end rule ubiquitylation. We think our system provides a general approach that can be extended to the identification of substrates of other E3 ligases.