Fission yeast 26S proteasome mutants are multi-drug resistant due to stabilization of the pap1 transcription factor

Here we report the result of a genetic screen for mutants resistant to the microtubule poison methyl benzimidazol-2-yl carbamate (MBC) that were also temperature sensitive for growth. In total the isolated mutants were distributed in ten complementation groups. Cloning experiments revealed that most of the mutants were in essential genes encoding various 26S proteasome subunits. We found that the proteasome mutants are multi-drug resistant due to stabilization of the stress-activated transcription factor Pap1. We show that the ubiquitylation and ultimately the degradation of Pap1 depend on the Rhp6/Ubc2 E2 ubiquitin conjugating enzyme and the Ubr1 E3 ubiquitin-protein ligase. Accordingly, mutants lacking Rhp6 or Ubr1 display drug-resistant phenotypes

The mts complementation groups.

<p>The mts complementation groups.</p

Stabilization of Pap1 in the mts mutants leads increased obr1⁺ expression.

<p>(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 <i>mts10-1</i> (<i>crm1</i>) mutant. A <i>pap1</i>Δ mutant was included as a control. (b) The degradation kinetics of GFP-tagged Pap1 was followed by blotting of wild type (wt) and <i>mts2-1</i> 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 <i>mts2-1</i> 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 <i>mts10-1</i> (<i>crm1</i>) mutant. No Obr1 was detected in the <i>pap1</i>Δ mutant.</p

Pap1 is ubiquitylated.

<p>To determine the ubiquitylation status of Pap1, wild type cells and as a control a <i>pap1</i>Δ 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 <i>pap1</i>Δ strain or in the vector control.</p

Fission yeast strains used in this study.

<p>Fission yeast strains used in this study.</p

The mts mutants are multi-drug resistant.

<p>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 <i>mts</i> mutants displayed resistance.</p