Figure 7.

Binding and ubiquitinylation of denatured luciferase by Ubr1. (A) Purified luciferase was kept native or denatured with guanidinium hydrochloride (GnHCl) before an ubiquitinylation reaction with Ubr1, Ube1, and UbcH2 and ³²P-labeled ubiquitin. Reactions were immunoprecipitated with anti-luciferase and were resolved on a denaturing gel before being visualized by autoradiography. Arrow denotes monoubiquitinylated luciferase. (B) Denatured luciferase was incubated with different components of the ubiquitinylation reaction as indicated. A RING mutant of Ubr1, MR1, was also used in this experiment. (C) Purified luciferase was preincubated at the indicated temperatures for 30 min before being split into two aliquots. One was added to an ubiquitinylation reaction containing Ubr1, Ube1 (E1), UbcH2 (E2), and ³²P-labeled ubiquitin (top panel). The second aliquot was used to measure remaining luciferase activity (graph in bottom panel). The bars represent luciferase activity as a percentage of the aliquot maintained at 4°C. (D) Purified luciferase was incubated with or without Ubr1 before denaturation at 42°C. The total reaction (T) was separated into supernatant (S) and pellet (P) fractions after centrifugation. The amount of luciferase in each fraction was determined by Western blot. (E) Luciferase was kept native or denatured at 42°C in the absence or presence of Ubr1. Ubr1 was immunoprecipitated, and the amount of luciferase that coimmunoprecipitated was determined by Western blot. (F) Ubiquitinylation of luciferase when Ubr1 is present during the denaturation step. Luciferase was denatured by incubation at 42°C by itself (lane 1) or in the presence of Ubr1 (lane 4) or HDM2 (lane 7). The luciferase was then added to reactions containing E1 and Ubc2 (lane 2); E1, Ubc2, and Ubr1 (lane 3); E1 and Ubc5 (lane 5); and E1, Ubc5, and HDM2 (lane 6). All reactions contained ATP and ubiquitin. Luciferase was visualized by Western blot. Arrow denotes nonubiquitinylated luciferase. (G) IP of Flag-Ubr1 and Western blot for coimmunoprecipitating luciferase before and after heat shock at 42°C for 30 min.