Identification and characterization of activating and conjugating enzymes of the ubiquitin system from the unicellular alga Chlamydomonas reinhardtii

Using a biochemical approach we identified families of ubiquitin-activating and ubiquitin-conjugating enzymes in Chlamydomonas reinhardtii. The family of ubiquitin-activating enzymes, characterized by their ability to form thioesters with ubiquitin and eluting off a ubiquitin affinity column by ATP-depletion probably consists of at least four members. Whereas one of these enzymes is active under a broad range of pH values, thioester of the other UBAs with ubiquitin is restricted to pH 7.5. Two ubiquitin-activating enzymes are metabolically phosphorylated which is assumed to be an activity control mechanism. Most of the 7 ubiquitin-conjugating enzymes detected in this study were found to bind rather tightly to an anion exchange column, and eluted off the column at specific salt concentrations. Two of the ubiquitin-conjugating enzymes described here did, however, not bind to this column. These enzymes can, as all other C. reinhardtii ubiquitin-conjugating enzymes, perform thioester formation with ubiquitin regardless of the source (plant/animal) of the ubiquitin-activating enzyme.