Identification of α-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 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.

again at the second meiotic metaphase until fertilization. The proteasomes are thought to be involved in regulating the maturation and fertilization of oocytes [6,7]. Previously we identified the proteasomal subunit modified during oocyte maturation in Xenopus and goldfish as α 4 [8,9]. In the present study, we cloned three unidentified αtype subunits of Xenopus and prepared antibodies for a total of five subunits. Using a set of specific antibodies, we analyzed changes in all α subunits composing the 26S proteasome during the meiotic cell cycle. We demonstrated that 6 of the subunits exist as a heterogeneous population in frog oocytes and identified another subunit in addition to α4 which was modified meiotic cell cycle dependently.

Isolation and characterization of cDNA clones
A BLAST search of the Xenopus EST database was conducted using known proteasomal subunit α cDNAs. From the data for each subunit, full-length ORFs were obtained by PCR. The amplified cDNAs were 741, 726 and 786 bp long. The clones encode proteins of 246, 241 and 261 amino acid residues with a predicted molecular mass of 27463, 26402 and 29327 daltons, respectively (Fig. 1). Comparison of the amino acid sequence revealed that these molecules are highly homologous to the α1, α5 and α6 subunits in humans (overall identity 91.5-95.4%) [10,11], Drosophila (53.2-69.1%) [12,13] and yeast (53.2-61.7%) [14][15][16] (Fig. 2). Thus, we concluded that the cDNAs isolated in this study encode the α1, α5 and α6 subunits of the Xenopus 20S proteasome. We named these clones α1_xl, α5_xl and α6_xl (α1, α5 and α6 subunits of Xenopus laevis) according to a systematic nomenclature [5]. Figure 2 represents a comparison of amino acid sequences predicted from cDNA sequences of α-type subunits of the Xenopus 20S proteasome. Overall identity between the subunits was 25.1-38.4 %. A consensus sequence for α-type proteasomal subunits was conserved. Interestingly, a conserved sequence for β-type proteasomal subunits was found in the α3 subunit [17].

Comparison of proteasomes purified from immature and mature oocytes
Polyclonal antibodies specific for five subunits (α1, α3, α5, α6, and α7) were raised against purified recombinant proteins. The specificity of the antibodies was examined by immunoblotting with the cytosol fraction and the purified 26S proteasome (Fig. 3). Each antibody preparation displayed a specific reaction for different polypeptides in both samples. Recombinant proteins from the cDNAs clearly cross-reacted with each antibody (data not shown). Thus, specific antibodies for each subunit were prepared. With these antibodies and previously described monoclonal antibodies for subunits α2 and α4 [18], changes to all α-type subunits during Xenopus meiotic maturation were analyzed. The modifications were demonstrated by 2D-PAGE (Fig. 4). The α7 subunit antibodies gave a single spot but all of the other antisera produced more than one spot, suggesting that the α1-α6 subunits undergo some type of modification in oocytes of Xenopus as demonstrated in other species [19,20]. A difference in the spots between the 26S proteasome from immature and mature oocytes was detected in the blots of subunits α2 and α4. In blots of α2 and α4, only a major spot was detected in the 26S proteasome from mature oocytes (in M-phase). It is suggested that the α4 subunit is phosphorylated in immature oocytes and dephosphorylated in mature oocytes [8]. Likewise, it is speculated that part of the α2 subunit is phosphorylated in interphase and dephosphorylated in metaphase. These results suggest that the subunits of 26S proteasomes are changed by meiotic cell cycle-dependent modifications. It can be speculated that these modifications are involved in the regulation of the meiotic cell cycle.
The modification of proteasomal subunits and factors interacting with proteasomes may be involved in the regulation of proteasome function [21]. By two-dimensional polyacrylamide gel electrophoresis, up to 20 different polypeptides were separated from the 20S proteasome which was shown to be composed of 14 gene products [22]. Furthermore, changes in proteasomal subunit composition under different physiological conditions and the likely existence of a different subpopulation of proteasomes have been reported [12,23]. All these results suggest that the subunit composition of proteasomes, and likely their activity, is under complex control in vivo. Some of these changes may be due to post-translational modifications of the proteasomal subunits. Regarding protein modification, there have been several reports about the phosphorylation of proteasomal subunits. Phosphorylated proteasomal subunits were detected in crude preparations from cultured Drosophila cells [22]. Several subunits of the 20S proteasome could be phosphorylated in vitro by a cyclic AMP-dependent protein kinase copurifying with the bovine pituitary 20S proteasome [24]. Castaño et al. [25] (1996) identified the CKII phosphorylating subunit and its phosphorylation sites as the C8 component (α7 subunit) and serine-243 and serine-250, respectively. CKII was also reported to phosphorylate the C2 component (α6 subunit) in rice [26]. The phosphorylation of subunits in the 26S proteasome in vivo was investigated using cultured human cells. Mason et al. [27] (1996) showed the phosphorylated subunits to be the C8 (α7 subunit) and C9 (α3 subunit) components in the 20S core, and the S4 (Rpt2p) subunit and several other components in regulatory particles [28]. Recent approaches have revealed post-translational modifications to many of the subunits. In the yeast 20S proteasome, the α2and α4subunits are phosphorylated at either a serine or Amino acid sequence comparison of the Xenopus, human, Drosophila, and Yeast α1, α5 and α6 proteasome subunits    threonine residue, and the α7-subunit is phosphorylated at tyrosine residue(s) [20]. In the human 20S proteasome, more than two spots were identified in all α-type subunits except α5 and phosphorylation of the α7-subunit at serine-250 was revealed [19]. However the sites and kinases responsible for the phosphorylation of the α2 and α4 sub-units of the 20S proteasome have yet to be demonstrated. The modification of these proteins is one possible mechanism regulating the functions of the 26S proteasome during the meiotic cell cycle. Consensus sequences for phosphorylation sites are conserved in these subunits [8,29]. Cyclic-AMP dependent protein kinase is responsi-Amino acid sequence comparison of the Xenopus proteasomal α subunits Figure 2 Amino acid sequence comparison of the Xenopus proteasomal α subunits. Matched sequences are boxed. The proteasomal α-type and β-type signatures were detemined by using the 'PROSITE' database [17] and are boxed. The numbers refer to the amino acid position at the end of each line. 1_xl ble for the G2/M and metaphase/anaphase transitions [30]. Calcium/calmodulin-dependent protein kinase II is shown to be involved in the exit from metaphase II arrest at fertilization in Xenopus [31]. It can be hypothesized that these kinases are involved in the regulation of 26S proteasome activity. The identification of kinases and the phosphorylation sites of the α2 and α4 subunits may reveal how the modification of proteasomal subunits is involved in controlling the cell cycle. Currently, we have identified one of the protein kinase for α4 subunit as Casein Kina-seIα [32]. Possible regulation of 26S proteasome activity by this kinase is under investigation.
Recently, alternative subunits of proteasomes have been identified. In Drosophila where alternative α-type, β-type and 19S cap subunits are expressed from separate genes during spermatogenesis [33] and in Arabidopsis and rice where alternative isoforms of most proteasome subunits are differentially expressed from separate genes during development [34,35]. There are also examples of alternative β-type subunits in mammals (e.g., γ-interferon inducible "immunoproteasome" subunits β1i, β2i and β5i) [36]. Alternative subunits have yet to be identified in Xenopus, there is a possibility that the changes in the spots identified in this study may derive from differential expression of alternative subunits from paralogous genes.
(2) Six subunits but not α7_XL are modified in immature oocytes in X. laevis.

Production of recombinant proteins and preparation of antibodies
The recombinant proteins were produced in E. coli BL21 (LysE) and purified by SDS-PAGE as described [6]. Polyclonal antibodies specific for each subunit were raised against purified recombinant proteins according to a procedure described before using guinea pigs [40]. Anti serums, which recognize the bands of each subunit, were obtained.