Characteristics of 26 S proteases from fission yeast mutants, which arrest in mitosis.
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.
Seeger
M
M
Institute of Biochemistry, Humboldt-University Medical Faculty (Charité), Berlin, Germany.
Gordon
C
C
Ferrell
K
K
Dubiel
W
W
D000255:Adenosine Triphosphate / Q000378:metabolism; D000705:Anaphase; D001081:Apyrase / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004789:Enzyme Activation; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D008938:Mitosis; D009097:Multienzyme Complexes / Q000378:metabolism; D009154:Mutation; D010447:Peptide Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D012568:Schizosaccharomyces / Q000201:enzymology*; D013696:Temperature; D014452:Ubiquitins / Q000378:metabolism
D009097:Multienzyme Complexes; D014452:Ubiquitins; D000255:Adenosine Triphosphate; D010447:Peptide Hydrolases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D001081:Apyrase