Table 1.
Fission yeast strains used in this study.
Table 2.
The mts complementation groups.
Figure 1.
The mts mutants are multi-drug resistant.
The indicated yeast strains (lower left panel) were streaked onto solid medium containing MBC, brefeldin A, staurosporine or caffeine at the shown concentrations and incubated for 48 hours at room temperature. On the control medium lacking drugs (upper left panel) all the strains grew. When the indicated drugs were added to the media the growth of wild type cells was compromised, while the mts mutants displayed resistance.
Figure 2.
Stabilization of Pap1 in the mts mutants leads increased obr1+ expression.
(a) To compare the steady state levels of Pap1 cell extracts of the indicated strains were prepared and analyzed by SDS-PAGE and Western blotting using antibodies to Pap1. Actin served as a loading control. Compared to wild type cells, the Pap1 levels were increased in the proteasome mutants, but not in the mts10-1 (crm1) mutant. A pap1Δ mutant was included as a control. (b) The degradation kinetics of GFP-tagged Pap1 was followed by blotting of wild type (wt) and mts2-1 cultures treated with cycloheximide (CHX). α-tubulin served as a loading control. In wild type cells Pap1 was rapidly degraded with a half-life of about 50 minutes. In the mts2-1 background Pap1 was stabilized (c) To compare the steady state levels of the Pap1 target Obr1 cell extracts of the indicated strains were prepared and analyzed by SDS-PAGE and Western blotting using antibodies to Obr1. Tubulin served as a loading control. Compared to wild type cells, the Obr1 levels were increased in the proteasome mutants and, as expected, in the mts10-1 (crm1) mutant. No Obr1 was detected in the pap1Δ mutant.
Figure 3.
The multi-drug resistance of the mts mutants depends on Pap1.
The indicated yeast strains (left panel) were streaked onto solid medium containing MBC 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 6 µg/mL MBC some growth of the wild type was still apparent, while no growth of the other strains was observed.
Figure 4.
To determine the ubiquitylation status of Pap1, wild type cells and as a control a pap1Δ strain were transformed to express 6His-tagged ubiquitin. The 6His-tagged ubiquitin was then precipitated using Ni2+ agarose under denaturing conditions in 8 M urea. The precipitates were then washed three times in a denaturing buffer and analyzed by Western blotting using antibodies to Pap1. Prior to precipitation, the protein concentrations were determined and normalized. In total 5 mg cell protein was used for each precipitation. Ubiquitylated species of Pap1 were detected in wild type cells expressing the 6His-tagged ubiquitin, but not in the pap1Δ strain or in the vector control.
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.