Identification of the Xenopus 20S proteasome alpha 4 subunit which is modified in the meiotic cell cycle

The proteasomes are large, multi-subunit particles that act as the proteoly tic machinery for most of the regulated intracellular protein degradation i n eukaryotic cells. To investigate the regulatory mechanism for the 26S pro teasome in cell-cycle events, we purified this proteasome from immature and mature oocytes, and compared its subunits. Immunoblot analysis of 26S prot easomes showed a difference in the subunit of the 20S proteasome. A monoclo nal 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 regulat ion of the meiotic cell cycle. Here we describe the molecular cloning of on e of the cl subunits of the 20S proteasome from a Xenopus ovarian cDNA libr ary using an anti-GC3 beta monoclonal antibody. From the screening, two typ es of cDNA are obtained, one 856 bp, the other 984 bp long. The deduced ami no-acid sequences comprise 247 and 248 residues, respectively. These deduce d amino-acid sequences are highly homologous to those of alpha 4 subunits o f other vertebrates. Phosphatase treatment of 26S proteasome revealed the u pper band to be a phosphorylated form of the lower band. These results sugg est 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) 1 999 Elsevier Science B.V. All rights reserved.