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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.
Fig. 1. Immunoblotting of the purified proteasomes. 20S and 26S proteasomes were electrophoresed under denaturing conditions (12.0% gel) and stained with Coomassie Brilliant Blue (CBBR), or immunostained with monoclonal antibodies (anti-GC4/5 and anti-GC3β) after electroblotting. A 31.5 kDa band which cross-reacted with anti-GC3β is indicated by an asterisk. Lanes I and M indicate proteasomes from immature and mature oocytes, respectively. Molecular masses of standard proteins are indicated at the left.
Fig. 2. 2D-PAGE analysis of the 26S proteasomes from immature and mature oocytes. The 26S proteasomes from immature (I) and mature (M) oocytes were subjected to 2D-PAGE followed by immunostaining by anti-Xenopus 20S proteasome polyclonal antibody (Tokumoto et al., 1999) (A), anti-GC3β (B) and a mixture of anti-GC4/5 and anti-GC3β (C). The spot of 31.5 kDa which cross-reacted with anti-GC3β is indicated by an asterisk.
Fig. 3. Nucleotide and deduced amino-acid sequences of the α4_xl genes of Xenopus. The upper letters correspond to the amino-acid sequence of α4_xl-A. Nucleotide sequences of the two genes are aligned with some gaps (hyphens) to give maximum matching. Identical nucleotides are indicated by dots. The numbers refer to the nucleotide positions at the end of each line. The positions used for design of the specific primers for RT–PCR (P1–P3) and SpeI site are underlined. The GenBank accession numbers for these sequences are AB004782 (α4_xl-A) and AB027463 (α4_xl-B).
Fig. 4. Amino-acid sequence comparison of the Xenopus, human, rat, and chicken α4 proteasome subunits. Matched sequences are boxed. Consensus sequences for calcium/calmodulin-dependent kinase II (CaMKII), cAMP/cGMP-dependent kinase (cAMP/cGMP), casein kinase II (CKII) and tyrosine kinase (TPK) are indicated. The numbers refer to the amino-acid position at the end of each line.
Fig. 5. Electrophoresis of RT–PCR products of α4_xl-A and α4_xl-B. Amplification of fragments of α4_xl-A and α4_xl-B was performed as described in Materials and methods, using total RNAs from embryos at indicated stages, unfertilized eggs (Egg) and each cDNA inserted into pBluescript SK (−) (Cont). Developmental stages of embryos were according to Niewkoop and Faber's (1967) developmental stages. Lanes A and B indicate the amplification by a pair of primers for α4_xl-A and α4_xl-B, respectively. To confirm the specific amplification, the PCR products were digested by SpeI. By SpeI digestion, the PCR products of α4_xl-A (602 bp) cleaved into two fragments (411 and 191 bp). PCR products of α4_xl-B possess no cutting site for SpeI. The PCR products before (panel A) and after (panel B) the SpeI digestion were electrophoresed on a 1.5% agarose gel and stained with ethidium bromide.
Fig. 6. Alkaline phosphatase treatment of 26S proteasome. Purified 26S proteasomes from immature oocytes (8 μg) were treated with (+) or without (−) calf intestine alkaline phosphatase as described in Materials and methods. (A) Immunoprecipitates were electrophoresed under denaturing conditions (12.0% gel) and immunostained with monoclonal antibody (anti-GC3β) after electroblotting. The 31.5 kDa band is indicated by an asterisk. (B) SDS-independent (SDS−) and -dependent (SDS+) Suc-LLVY-MCA hydrolyzing activity of 26S proteasomes treated with (+) or without (−) alkaline phosphatase was determined. Activities are indicated as a percentage of the activity of control experiment.