Identification of alpha-type subunits of the Xenopus 20S proteasome and analysis of their changes during the meiotic cell cycle.
BACKGROUND
The 26S proteasome is the proteolytic machinery of the ubiquitin-dependent proteolytic system responsible for most of the regulated intracellular protein degradation in eukaryotic cells. Previously, we demonstrated meiotic cell cycle dependent phosphorylation of alpha4 subunit of the 26S proteasome. In this study, we analyzed the changes in the spotting pattern separated by 2-D gel electrophoresis of alpha subunits during Xenopus oocyte maturation.


RESULTS
We identified cDNA for three alpha-type subunits (alpha1, alpha5 and alpha6) of Xenopus, then prepared antibodies specific for five subunits (alpha1, alpha3, alpha5, alpha6, and alpha7). With these antibodies and previously described monoclonal antibodies for subunits alpha2 and alpha4, modifications to all alpha-type subunits of the 26S proteasome during Xenopus meiotic maturation were examined by 2D-PAGE. More than one spot for all subunits except alpha7 was identified. Immunoblot analysis of 26S proteasomes purified from immature and mature oocytes showed a difference in the blots of alpha2 and alpha4, with an additional spot detected in the 26S proteasome from immature oocytes (in G2-phase).


CONCLUSIONS
Six of alpha-type subunits of the Xenopus 26S proteasome are modified in Xenopus immature oocytes and two subunits (alpha2 and alpha4) are modified meiotic cell cycle-dependently.
Wakata
Yuka
Y
Department of Biology and Geosciences, Faculty of Science, National University Corporation Shizuoka University, Shizuoka 422-8529, Japan. r0335030@ipc.shizuoka.ac.jp <r0335030@ipc.shizuoka.ac.jp>
Tokumoto
Mika
M
Horiguchi
Ryo
R
Ishikawa
Katsutoshi
K
Nagahama
Yoshitaka
Y
Tokumoto
Toshinobu
T
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002453:Cell Cycle; D002454:Cell Differentiation; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D018076:DNA, Complementary / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D015151:Immunoblotting; D008540:Meiosis; D008969:Molecular Sequence Data; D009865:Oocytes / Q000166:cytology* / Q000201:enzymology / Q000254:growth & development / Q000276:immunology; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014981:Xenopus / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D021122:Protein Subunits; D046988:Proteasome Endopeptidase Complex