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Characteristics of 26 S proteases from fission yeast mutants, which arrest in mitosis
M Seeger1, C Gordon, K Ferrell
1Institute of Biochemistry, Humboldt-University Medical Faculty (Charité), Berlin, Germany.
Abstract:
We have isolated the 26 S protease from the fission yeast Schizosaccharomyces pombe. The affinity-purified enzyme contains the two regulatory ATPases mts2+, a homolog of human S4, and CIM5, a homolog of human MSS1 = S7. We show that mts3+, a homolog of the budding yeast NIN1 protein and human S14, is a true component of the 19 S regulatory complex from the fission yeast. The 26 S proteases purified from two thermosensitive mutants, mts2-1 and mts3-1, which arrest in cell cycle at the restrictive temperature (37 degrees C), have been compared with the wild-type enzyme after growing cells at permissive (25 degrees C) and non-permissive temperatures. We demonstrate that mutated mts2 protein is integrated into the protease complex prepared from mts2 cells, whereas mutated mts3 is not present in the 19 S regulatory complex from mts3 cells. The two mutant 26 S proteases isolated after growing cells at 37 degrees C remain stable for two hours at 37 degrees C as measured by ATP-dependent cleavage of the fluorogenic peptide sucLLVY-MCA. At the restrictive temperature, the mutant 26 S proteases do not degrade ubiquitin-[125I]lysozyme conjugates in an ATP-dependent manner, indicating that mts2+ and mts3+ are essential for ubiquitin conjugate degradation. This explains the conditional lethality of the mutants and the cell-cycle arrest in metaphase to anaphase transition. In addition, our data demonstrate that the ATPases of the 26 S enzyme are not redundant.
Insights
The fission yeast 26S proteasome requires mts2+ and mts3+ proteins for ubiquitin conjugate degradation, explaining cell cycle arrest. These ATPases are essential and not redundant for proteasome function.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The 26S proteasome is a crucial multi-subunit protease complex responsible for protein degradation in eukaryotic cells.
- Components of the 26S proteasome, including regulatory ATPases, are conserved across species, highlighting their fundamental biological roles.
- Understanding the function of specific proteasome subunits is key to deciphering cellular processes like cell cycle regulation.
Purpose of the Study:
- To investigate the roles of mts2+ and mts3+ proteins in the function of the 26S proteasome in fission yeast, Schizosaccharomyces pombe.
- To determine the contribution of these subunits to the degradation of ubiquitinated proteins and their impact on cell cycle progression.
- To elucidate the structural integration and functional necessity of mts2+ and mts3+ within the 19S regulatory complex.
Main Methods:
- Affinity purification of the 26S proteasome from wild-type and thermosensitive mutant strains (mts2-1, mts3-1) of Schizosaccharomyces pombe.
- Biochemical assays to assess the ATP-dependent cleavage activity and degradation of ubiquitin-[125I]lysozyme conjugates.
- Analysis of the composition of the 19S regulatory complex in mutant proteasomes under permissive and restrictive temperature conditions.
Main Results:
- The 26S proteasome isolated from fission yeast contains mts2+ and CIM5, with mts3+ confirmed as a component of the 19S regulatory complex.
- Mutated mts2 protein was incorporated into the proteasome, but mutated mts3 was absent from the 19S complex in respective mutants.
- Mutant 26S proteasomes showed impaired ATP-dependent degradation of ubiquitin conjugates at the restrictive temperature, leading to cell cycle arrest.
Conclusions:
- The mts2+ and mts3+ proteins are essential for the ubiquitin conjugate degradation activity of the 26S proteasome in fission yeast.
- The differential incorporation of mutated subunits suggests distinct roles in the structural integrity and function of the 19S regulatory complex.
- These findings explain the conditional lethality and cell cycle arrest observed in mts2 and mts3 mutants, underscoring the non-redundant functions of these ATPases.