Characteristics of 26 S Proteases from Fission Yeast Mutants, which Arrest in Mitosis

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°C), have been compared with the wild-type enzyme after growing cells at permissive (25°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°C remain stable for two hours at 37°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, indicat ing 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.