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 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.
Tokumoto
M
M
Laboratory of Reproductive Biology, National Institute for Basic Biology, Okazaki, Japan. sbttoku@ipc.shizuoka.ac.jp
Horiguchi
R
R
Nagahama
Y
Y
Ishikawa
K
K
Tokumoto
T
T
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
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D009097:Multienzyme Complexes; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D018833:Chaperonins