Identification of the Xenopus 20S proteasome α4 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, GC3 beta, 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 cl subunits of the 20S proteasome from a Xenopus ovarian cDNA library using an anti-GC3 beta monoclonal antibody. From the screening, two types of cDNA are obtained, one 856 bp, the other 984 bp long. The deduced amino-acid sequences comprise 247 and 248 residues, respectively. These deduced amino-acid sequences are highly homologous to those of alpha 4 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 alpha 4 subunit of the Xenopus 20S proteasome, alpha 4_xl, is phosphorylated in G2-phase and dephosphorylated in M-phase. (C) 1999 Elsevier Science B.V. All rights reserved.