title: Two proteins, a goldfish 20S proteasome subunit and the protein interacting with 26S proteasome, change in the meiotic cell cycle.
abstract: To investigate the regulatory mechanism for the proteasome in the meiotic cell cycle, we purified the 26S proteasome from immature (in G2-phase) and mature (in M-phase) oocytes, and compared its subunits by immunoblotting. At least two protein bands, at 30 kDa (detected by GC3beta antibody) and 62 kDa (detected by 1-4D5 antibody), differed between 26S proteasomes. A monoclonal antibody, GC3beta cross-reacted with two bands in the 26S proteasome from immature oocytes, however, the upper band was absent in the 26S proteasome from mature oocytes. The 62-kDa protein band detected by 1-4D5 antibody was not detected in the immature oocyte 26S proteasome; however, a band was detected in mature oocyte 26S proteasome. The cDNAs encoding these proteins were isolated by an immunoscreening method using the monoclonal antibodies. The 30-kDa protein was an alpha4 subunit, which is one of the alpha-subunit group of the 20S proteasome, and the 62-kDa protein was a homologue of CCTepsilon, one of the components of eukaryotic molecular chaperones. Phosphatase treatment of the 26S proteasome revealed that a part of the alpha4 subunit of goldfish 20S proteasome, alpha4_ca, is phosphorylated in G2-phase and dephosphorylated in M-phase. A binding assay using a recombinant goldfish CCTepsilon revealed that unmodified CCTepsilon interacts with the 26S proteasome. Fertilization triggers a transition from meiotic metaphase to mitotic interphase. During fertilization, a GC3beta cross-reacting upper band reappeared. The 62-kDa band dissociated from the 26S proteasome. As a result, the 26S proteasome changed to an immature type from a mature type during fertilization. These results suggest that the 26S proteasome is changed reversibly during the meiotic cell cycle by modification of its subunits and interactions between regulators.
authors.affiliation: Laboratory of Reproductive Biology, National Institute for Basic Biology, Okazaki, Japan. sbttoku@ipc.shizuoka.ac.jp
authors.forename: M
authors.initials: M
authors.lastname: Tokumoto
authors.forename: R
authors.initials: R
authors.lastname: Horiguchi
authors.forename: Y
authors.initials: Y
authors.lastname: Nagahama
authors.forename: K
authors.initials: K
authors.lastname: Ishikawa
authors.forename: T
authors.initials: T
authors.lastname: Tokumoto
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D018833:Chaperonins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D003429:Cross Reactions / Q000276:immunology; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D005306:Fertilization; D016195:G2 Phase; D006054:Goldfish; D006801:Humans; D008540:Meiosis; D008938:Mitosis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D009865:Oocytes / Q000166:cytology / Q000201:enzymology / Q000254:growth & development / Q000378:metabolism*; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D016415:Sequence Alignment
chemical_list: D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D009097:Multienzyme Complexes; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D018833:Chaperonins