Identification of α-type subunits of the Xenopus 20S proteasome and analysis of their changes during the meiotic cell cycle

Yuka Wakata¹ , Mika Tokumoto¹^,2 , Ryo Horiguchi¹^,2 , Katsutoshi Ishikawa¹ , Yoshitaka Nagahama²^,3 and Toshinobu Tokumoto¹^,2

¹Department of Biology and Geosciences, Faculty of Science, National University Corporation Shizuoka University, Shizuoka 422-8529, Japan

²CREST Research Project, Japan Science and Technology Corporation, Japan

³Laboratory of Reproductive Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan

author email corresponding author email

BMC Biochemistry 2004, 5:18doi:10.1186/1471-2091-5-18


Published:	17 December 2004

Abstract

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 α4 subunit of the 26S proteasome. In this study, we analyzed the changes in the spotting pattern separated by 2-D gel electrophoresis of α subunits during Xenopus oocyte maturation.

Results

We identified cDNA for three α-type subunits (α1, α5 and α6) of Xenopus, then prepared antibodies specific for five subunits (α1, α3, α5, α6, and α7). With these antibodies and previously described monoclonal antibodies for subunits α2 and α4, modifications to all α-type subunits of the 26S proteasome during Xenopus meiotic maturation were examined by 2D-PAGE. More than one spot for all subunits except α7 was identified. Immunoblot analysis of 26S proteasomes purified from immature and mature oocytes showed a difference in the blots of α2 and α4, with an additional spot detected in the 26S proteasome from immature oocytes (in G2-phase).

Conclusions

Six of α-type subunits of the Xenopus 26S proteasome are modified in Xenopus immature oocytes and two subunits (α2 and α4) are modified meiotic cell cycle-dependently.