Identification of the Xenopus 20S proteasome alpha4 subunit which is modified in the meiotic cell cycle.
The proteasomes are large, multi-subunit particles that act as the proteolytic machinery for most of the regulated intracellular protein degradation in eukaryotic cells. To investigate the regulatory mechanism for the 26S proteasome in cell-cycle events, we purified this proteasome from immature and mature oocytes, and compared its subunits. Immunoblot analysis of 26S proteasomes showed a difference in the subunit of the 20S proteasome. A monoclonal antibody, GC3beta, cross-reacted with two bands in the 26S proteasome from immature oocytes (in G2-phase); however, the upper band was absent in the 26S proteasome from mature oocytes (in M-phase). These results suggest that changes in the subunits of 26S proteasomes are involved in the regulation of the meiotic cell cycle. Here we describe the molecular cloning of one of the alpha subunits of the 20S proteasome from a Xenopus ovarian cDNA library using an anti-GC3beta monoclonal antibody. From the screening, two types of cDNA are obtained, one 856bp, the other 984bp long. The deduced amino-acid sequences comprise 247 and 248 residues, respectively. These deduced amino-acid sequences are highly homologous to those of alpha4 subunits of other vertebrates. Phosphatase treatment of 26S proteasome revealed the upper band to be a phosphorylated form of the lower band. These results suggest that a part of the alpha4 subunit of the Xenopus 20S proteasome, alpha4_xl, is phosphorylated in G2-phase and dephosphorylated in M-phase.
Tokumoto
M
M
Laboratory of Reproductive Biology, National Institute for Basic Biology, Okazaki, Japan.
Horiguchi
R
R
Nagahama
Y
Y
Tokumoto
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002453:Cell Cycle / Q000235:genetics; D003546:Cysteine Endopeptidases / Q000032:analysis / Q000235:genetics*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D005260:Female; D008540:Meiosis / Q000235:genetics*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000032:analysis / Q000235:genetics*; D009865:Oocytes / Q000201:enzymology / Q000254:growth & development; D010447:Peptide Hydrolases / Q000032:analysis; D046988:Proteasome Endopeptidase Complex; D020033:Protein Isoforms / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis / Q000235:genetics*
D018076:DNA, Complementary; D009097:Multienzyme Complexes; D020033:Protein Isoforms; D010447:Peptide Hydrolases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease