Figure 5. Pap1 is ubiquitylated by Rhp6/Ubc2 and Ubr1.

(a) To identify which E2 ubiquitin conjugating enzyme is responsible for ubiquitylating Pap1, the indicated E2 null mutants were streaked onto solid medium containing MBC and grown for 48 hours at room temperature. Since the rhp6Δ cells grow in the presence of MBC (arrowhead) it was a candidate E2 in Pap1 ubiquitylation. (b) To identify the E2 and E3 enzymes responsible for ubiquitylating Pap1, the indicated strains were transformed to express 6His-tagged ubiquitin. The 6His-tagged ubiquitin was then precipitated using Ni²⁺ agarose under denaturing conditions and analyzed by Western blotting using antibodies to Pap1. Ubiquitylated species of Pap1 were detected in wild type, ubr11Δ and rhp18Δ cells, but not in cells lacking the E2 ubiquitin conjugating enzyme Rhp6 and in cells lacking the E3 ubiquitin-protein ligase Ubr1. (c) The indicated yeast strains (left panel) were streaked onto solid medium containing MBC or caffeine at the shown concentrations and incubated for 48 hours at room temperature. On the control medium lacking drugs all the strains grew. In the presence of drugs the growth of wild type cells was compromised, while the ubr1Δ mutant displayed resistance.