BACKGROUND
eukaryotic cells, from yeast to human, contain large nonlysosomal proteases called proteasomes  <cit> .
the 26s proteasome is part of the ubiquitin-dependent proteolytic system, which regulates proteins through a mechanism of selective degradation  <cit> .
the 26s proteasome is composed of a 20s proteasome as a catalytic core and regulatory particles at either end.
the subunits of the 20s proteasome subunits can be classified into two families, α and β. in eukaryotes, the 20s proteasome contains seven α-type subunits and seven β-type subunits.
the fourteen kinds of subunits are arranged in four rings of seven subunits and form an α7β7β7α <dig> structure  <cit> .
fully grown frog oocytes arrest in the late g <dig> phase of meiosis.
maturation-inducing hormone  acts on the oocytes, inducing final maturation and triggering germinal vesicle breakdown , and the oocytes arrest again at the second meiotic metaphase until fertilization.
the proteasomes are thought to be involved in regulating the maturation and fertilization of oocytes  <cit> .
previously we identified the proteasomal subunit modified during oocyte maturation in xenopus and goldfish as α  <dig>  <cit> .
in the present study, we cloned three unidentified α-type 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  <dig> of the subunits exist as a heterogeneous population in frog oocytes and identified another subunit in addition to α <dig> which was modified meiotic cell cycle dependently.
RESULTS
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  <dig>   <dig> and  <dig> bp long.
the clones encode proteins of  <dig>   <dig> and  <dig> amino acid residues with a predicted molecular mass of  <dig>   <dig> and  <dig> daltons, respectively .
comparison of the amino acid sequence revealed that these molecules are highly homologous to the α <dig>  α <dig> and α <dig> subunits in humans   <cit> , drosophila   <cit>  and yeast   <cit>  .
thus, we concluded that the cdnas isolated in this study encode the α <dig>  α <dig> and α <dig> subunits of the xenopus 20s proteasome.
we named these clones α1_xl, α5_xl and α6_xl  according to a systematic nomenclature  <cit> .
figure  <dig> 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  <dig> – <dig>  %.
a consensus sequence for α-type proteasomal subunits was conserved.
interestingly, a conserved sequence for β-type proteasomal subunits was found in the α <dig> subunit  <cit> .
comparison of proteasomes purified from immature and mature oocytes
polyclonal antibodies specific for five subunits  were raised against purified recombinant proteins.
the specificity of the antibodies was examined by immunoblotting with the cytosol fraction and the purified 26s proteasome .
each antibody preparation displayed a specific reaction for different polypeptides in both samples.
recombinant proteins from the cdnas clearly cross-reacted with each antibody .
thus, specific antibodies for each subunit were prepared.
with these antibodies and previously described monoclonal antibodies for subunits α <dig> and α <dig>  <cit> , changes to all α-type subunits during xenopus meiotic maturation were analyzed.
the modifications were demonstrated by 2d-page .
the α <dig> subunit antibodies gave a single spot but all of the other antisera produced more than one spot, suggesting that the α1–α <dig> subunits undergo some type of modification in oocytes of xenopus as demonstrated in other species  <cit> .
a difference in the spots between the 26s proteasome from immature and mature oocytes was detected in the blots of subunits α <dig> and α <dig>  in blots of α <dig> and α <dig>  only a major spot was detected in the 26s proteasome from mature oocytes .
it is suggested that the α <dig> subunit is phosphorylated in immature oocytes and dephosphorylated in mature oocytes  <cit> .
likewise, it is speculated that part of the α <dig> 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  <cit> .
by two-dimensional polyacrylamide gel electrophoresis, up to  <dig> different polypeptides were separated from the 20s proteasome which was shown to be composed of  <dig> gene products  <cit> .
furthermore, changes in proteasomal subunit composition under different physiological conditions and the likely existence of a different subpopulation of proteasomes have been reported  <cit> .
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  <cit> .
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  <cit> .
castaño et al.
<cit>   identified the ckii phosphorylating subunit and its phosphorylation sites as the c <dig> component  and serine- <dig> and serine- <dig>  respectively.
ckii was also reported to phosphorylate the c <dig> component  in rice  <cit> .
the phosphorylation of subunits in the 26s proteasome in vivo was investigated using cultured human cells.
mason et al.
<cit>   showed the phosphorylated subunits to be the c <dig>  and c <dig>  components in the 20s core, and the s <dig>  subunit and several other components in regulatory particles  <cit> .
recent approaches have revealed post-translational modifications to many of the subunits.
in the yeast 20s proteasome, the α2- and α4-subunits are phosphorylated at either a serine or threonine residue, and the α7-subunit is phosphorylated at tyrosine residue  <cit> .
in the human 20s proteasome, more than two spots were identified in all α-type subunits except α <dig> and phosphorylation of the α7-subunit at serine- <dig> was revealed  <cit> .
however the sites and kinases responsible for the phosphorylation of the α <dig> and α <dig> subunits 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  <cit> .
cyclic-amp dependent protein kinase is responsible for the g2/m and metaphase/anaphase transitions  <cit> .
calcium/calmodulin-dependent protein kinase ii is shown to be involved in the exit from metaphase ii arrest at fertilization in xenopus  <cit> .
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 α <dig> and α <dig> 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 α <dig> subunit as casein kinaseiα  <cit> .
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  <cit>  and in arabidopsis and rice where alternative isoforms of most proteasome subunits are differentially expressed from separate genes during development  <cit> .
there are also examples of alternative β-type subunits in mammals   <cit> .
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.
CONCLUSIONS
cdnas for three α-type proteasome subunits  of x. laevis were identified.
six subunits but not α7_xl are modified in immature oocytes in x. laevis.
α <dig>  α4_xls are modified during the meiotic cell cycle in x. laevis.

 eukaryotic cells, from yeast to human, contain large nonlysosomal proteases called proteasomes. the 26s proteasome is part of the ubiquitin-dependent proteolytic system, which regulates proteins through a mechanism of selective degradation. the 26s proteasome is composed of a 20s proteasome as a catalytic core and regulatory particles at either end. the subunits of the 20s proteasome subunits can be classified into two families, α and β. in eukaryotes, the 20s proteasome contains seven α-type subunits and seven β-type subunits. the fourteen kinds of subunits are arranged in four rings of seven subunits and form an α7β7β7α structure. fully grown frog oocytes arrest in the late g phase of meiosis. maturation-inducing hormone acts on the oocytes, inducing final maturation and triggering germinal vesicle breakdown, and the oocytes arrest again at the second meiotic metaphase until fertilization. the proteasomes are thought to be involved in regulating the maturation and fertilization of oocytes. previously we identified the proteasomal subunit modified during oocyte maturation in xenopus and goldfish as α. in the present study, we cloned three unidentified α-type 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 of the subunits exist as a heterogeneous population in frog oocytes and identified another subunit in addition to α 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 and bp long. the clones encode proteins of and amino acid residues with a predicted molecular mass of and daltons, respectively. comparison of the amino acid sequence revealed that these molecules are highly homologous to the α α and α subunits in humans, drosophila and yeast. thus, we concluded that the cdnas isolated in this study encode the α α and α subunits of the xenopus 20s proteasome. we named these clones α1_xl, α5_xl and α6_xl according to a systematic nomenclature. figure 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 – %. a consensus sequence for α-type proteasomal subunits was conserved. interestingly, a conserved sequence for β-type proteasomal subunits was found in the α subunit. comparison of proteasomes purified from immature and mature oocytes polyclonal antibodies specific for five subunits were raised against purified recombinant proteins. the specificity of the antibodies was examined by immunoblotting with the cytosol fraction and the purified 26s proteasome. each antibody preparation displayed a specific reaction for different polypeptides in both samples. recombinant proteins from the cdnas clearly cross-reacted with each antibody. thus, specific antibodies for each subunit were prepared. with these antibodies and previously described monoclonal antibodies for subunits α and α, changes to all α-type subunits during xenopus meiotic maturation were analyzed. the modifications were demonstrated by 2d-page. the α subunit antibodies gave a single spot but all of the other antisera produced more than one spot, suggesting that the α1–α subunits undergo some type of modification in oocytes of xenopus as demonstrated in other species. a difference in the spots between the 26s proteasome from immature and mature oocytes was detected in the blots of subunits α and α in blots of α and α only a major spot was detected in the 26s proteasome from mature oocytes. it is suggested that the α subunit is phosphorylated in immature oocytes and dephosphorylated in mature oocytes. likewise, it is speculated that part of the α 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. by two-dimensional polyacrylamide gel electrophoresis, up to different polypeptides were separated from the 20s proteasome which was shown to be composed of gene products. furthermore, changes in proteasomal subunit composition under different physiological conditions and the likely existence of a different subpopulation of proteasomes have been reported. 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. 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. castaño et al. identified the ckii phosphorylating subunit and its phosphorylation sites as the c component and serine- and serine- respectively. ckii was also reported to phosphorylate the c component in rice. the phosphorylation of subunits in the 26s proteasome in vivo was investigated using cultured human cells. mason et al. showed the phosphorylated subunits to be the c and c components in the 20s core, and the s subunit and several other components in regulatory particles. recent approaches have revealed post-translational modifications to many of the subunits. in the yeast 20s proteasome, the α2- and α4-subunits are phosphorylated at either a serine or threonine residue, and the α7-subunit is phosphorylated at tyrosine residue. in the human 20s proteasome, more than two spots were identified in all α-type subunits except α and phosphorylation of the α7-subunit at serine- was revealed. however the sites and kinases responsible for the phosphorylation of the α and α subunits 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. cyclic-amp dependent protein kinase is responsible for the g2/m and metaphase/anaphase transitions. calcium/calmodulin-dependent protein kinase ii is shown to be involved in the exit from metaphase ii arrest at fertilization in xenopus. 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 α and α 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 α subunit as casein kinaseiα. 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 and in arabidopsis and rice where alternative isoforms of most proteasome subunits are differentially expressed from separate genes during development. there are also examples of alternative β-type subunits in mammals. 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.  cdnas for three α-type proteasome subunits of x. laevis were identified. six subunits but not α7_xl are modified in immature oocytes in x. laevis. α α4_xls are modified during the meiotic cell cycle in x. laevis. 
immunoblot analysis of 26s proteasomes purified from immature and mature oocytes showed a difference in the blots of α and α with an additional spot detected in the 26s proteasome from immature oocytes. six of α-type subunits of the xenopus 26s proteasome are modified in xenopus immature oocytes and two subunits are modified meiotic cell cycle-dependently. with these antibodies and previously described monoclonal antibodies for subunits α and α modifications to all α-type subunits of the 26s proteasome during xenopus meiotic maturation were examined by 2d-page. in this study, we analyzed the changes in the spotting pattern separated by 2-d gel electrophoresis of α subunits during xenopus oocyte maturation. previously, we demonstrated meiotic cell cycle dependent phosphorylation of α subunit of the 26s proteasome. we identified cdna for three α-type subunits of xenopus, then prepared antibodies specific for five subunits. 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.
 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 α 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.  we identified cdna for three α-type subunits of xenopus, then prepared antibodies specific for five subunits. with these antibodies and previously described monoclonal antibodies for subunits α and α 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 α was identified. immunoblot analysis of 26s proteasomes purified from immature and mature oocytes showed a difference in the blots of α and α with an additional spot detected in the 26s proteasome from immature oocytes.  six of α-type subunits of the xenopus 26s proteasome are modified in xenopus immature oocytes and two subunits are modified meiotic cell cycle-dependently. 