Two proteins, a goldfish 20S proteasome subunit and the protein interacting with 26S proteasome, change in the meiotic cell cycle

To investigate the regulatory mechanism for the proteasome in the meiotic c ell cycle, we purified the 26S proteasome from immature (in G2-phase) and m ature (in M-phase) oocytes, and compared its subunits by immunoblotting. At least two protein bands, at 30 kDa (detected by GC3 beta antibody) and 62 kDa (detected by 1-4D5 antibody), differed between 26S proteasomes. A monoc lonal antibody, GC3 beta cross-reacted with two bands in the 26S proteasome from immature oocytes, however, the upper band was absent in the 26S prote asome from mature oocytes. The 62-kDa protein band detected by 1-4D5 antibo dy was not detected in the immature oocyte 26S proteasome; however, a band was detected in mature oocyte 26S proteasome. The cDNAs encoding these prot eins were isolated by an immunoscreening method using the monoclonal antibo dies. The 30-kDa protein was an alpha 4 subunit, which is one of the alpha- subunit group of the 20S proteasome, and the 62-kDa protein was a homologue of CCT epsilon, one of the components of eukaryotic molecular chaperones. Phosphatase treatment of the 26S proteasome revealed that a part of the alp ha 4 subunit of goldfish 20S proteasome, alpha 4_ca, is phosphorylated in G 2-phase and dephosphorylated in M-phase. A binding assay using a recombinan t goldfish CCT epsilon revealed that unmodified CCT epsilon interacts with the 26S proteasome. Fertilization triggers a transition from meiotic metaphase to mitotic inter phase. During fertilization, a GC3 beta cross-reacting upper band reappeare d. The 62-kDa band dissociated from the 26S proteasome. As a result, the 26 S proteasome changed to an immature type from a mature type during fertiliz ation. These results suggest that the 26S proteasome is changed reversibly during the meiotic cell cycle by modification of its subunits and interacti ons between regulators.