Figure 5.
In vitro ubiquitination of Cdc6p is dependent on Cdc4p. 35S-Labeled Cdc6p was prepared by in vitro translation as described in MATERIALS AND METHODS and used as a substrate for in vitro ubiquitination reactions. Lane 1 contains the input 35S-labeled Cdc6p without extract. Extract prepared from RJD885 cells (wild type for ubiquitination components) was used in the ubiquitination reactions in lanes 2–6. Extract from RJD893 cells (cdc4 and isogenic to RJD885) was used in reactions in lanes 7–12. The complete ubiquitination reaction (lanes 5 and 10) included DEAE-fractionated extract of G1-arrested cells, purified Cdc34p, insect cell lysate containing Clb5p/Cdc28p kinase, an ATP-regenerating system, salts, and ubiquitin. The reactions in lanes 6, 11, and 12 were supplemented with recombinant Cdc4p purified from insect cells. Cdc34p and Clb5p/Cdc28p were omitted where indicated.