|Budget Amount *help
¥8,400,000 (Direct Cost: ¥8,400,000)
Fiscal Year 1997: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1996: ¥6,900,000 (Direct Cost: ¥6,900,000)
1. Multiubiquitinated proteins were isolated from tempetature-sensitive nin1 mutant of budding yeast, which had been cultured at non-permissive temperature, by a procedure including Superose 6 gel filtration, FK2 antibody-agarose chromatography, and Mono Q FPLC.The isolated proteins were digested with lysylendopeptidase, and the digests were separated by reverse-phase HPLC,followed by amino acid sequence determination. By homology search, we propose that several proteins including porin are ubiquitinated.
2. In cell-free extract of Xenopus eggs, proteasome inhibitors caused a delay of cell cycle progression and accumulation of multiubiquitinated proteins. The accumulated proteins were also isolated by the procedure described above.
3. In heat-shock treated HeLa cells, multiubiquitinated proteins were accumulated in cytosol, while ubiquitinated histone H2A in nucleus was decreased.
4. By enzymeimmunoassay for multiubiquitin chains, it was found that serum concentration of multiubiquitin chains was higher in some patients than in healthy subjects.
5. Multiubiquitinated proteins were accumulated in temperature-sensitive sen3, nasl, and sun2 mutants, indicating that these components of the 26S proteasome play essential roles in degradation of multiubiquitinated proteins by the 26S proteasome.