Lub1 Participates in Ubiquitin Homeostasis and Stress Response via Maintenance of Cellular Ubiquitin Contents in Fission Yeast

Abstract

Ubiquitin-dependent proteolysis plays a pivotal role in stress responses. To investigate the mechanisms of these cellular processes, we have been studying Schizosaccharomyces pombe mutants that have altered sensitivities to various stress conditions. Here, we showed that Lub1, a homologue of Ufd3p/Zzz4p/Doa1p in budding yeast, is involved in the regulation of ubiquitin contents. Disruption of the lub1 ؉ gene resulted in monoubiquitin as well as multiubiquitin depletion without change in mRNA level and in hypersensitivity to various stress conditions. Consistently, overexpression of genes encoding ubiquitin suppressed the defects associated with lub1 mutation, indicating that the phenotypes of the lub1 mutants under stress conditions were due to cellular ubiquitin shortage at the posttranscriptional level. In addition, the lub1-deleted cells showed aberrant functions in ubiquitin/proteasome-dependent proteolysis, with accelerated degradation of ubiquitin. Also Cdc48, a stress-induced chaperon-like essential ATPase, was found to interact with Lub1, and this association might contribute to the stabilization of Lub1. Our results indicated that Lub1 is responsible for ubiquitin homeostasis at the protein level through a negative regulation of ubiquitin degradation.

DOA1/UFD3 Plays a Role in Sorting Ubiquitinated Membrane Proteins into Multivesicular Bodies

“…We next examined Ub levels within these transformants by immunoblotting cell extracts with anti-Ub antibodies and examining the level of Ub conjugates. Consistent with previous studies, loss of Doa1 significantly reduced the level of Ub (21,27,31). Using our particular lysis protocol, we found very little unconjugated Ub overall, making the comparison of Ub conjugates more informative for assessing the overall level of Ub.…”

“…Other analysis has shown that loss of Doa1 dramatically decreases Ub levels (21). Many of the phenotypes of doa1⌬ mutants can be suppressed by overexpressing Ub, suggesting that many of the noted doa1⌬ defects are solely an indirect consequence of lowered Ub levels (21,26,28,31). It is not presently clear how Ub is depleted in the absence of Doa1; however, inhibiting delivery and degradation of ubiquitinated proteins to the proteasome suppresses doa1⌬ growth defects, suggesting that more Ub may be degraded by the proteasome in the absence of Doa1 (24,28,31).…”

WD40 Repeat Propellers Define a Ubiquitin-Binding Domain that Regulates Turnover of F Box Proteins

“…Doa1 is a cofactor for the AAA ATPase Cdc48/p97, which helps convey ubiquitinated proteins to the Proteasome (Raasi and Wolf, 2007). Loss of Doa1 causes many cellular defects, including sensitivity to a wide range of drugs and elevated temperature, impaired sorting of ubiquitinated proteins to lysosomes/vacuoles, and defects in DNA repair (Ghislain et al, 1996; Lis and Romesberg, 2006; Ogiso et al, 2004; Ren et al, 2008). Steady-state levels of Ub are decreased in doa1 Δ null mutants, suggesting that Doa1 plays a role in maintaining Ub homeostasis (Ghislain et al, 1996; Mullally et al, 2006; Ren et al, 2008).…”

The Role of the Regulatory Subunit of Fission Yeast Calcineurin for in Vivo Activity and Its Relevance to FK506 Sensitivity

“…Cells were resuspended in ice-cold homogenizing buffer and homogenized using Mini-Beadbeater as described above. FLAG-Prz1 was immunoprecipitated with an agarose-immobilized anti-FLAG antibody as described previously (27). The immunoprecipitates were washed three times with 50 mM HEPES (pH 7.5) containing 150 mM NaCl, 10% glycerol, and 0.1% Triton X-100.…”