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Regulated interaction between polypeptide chain elongation factor-1 complex with the 26S proteasome during Xenopus oocyte maturation.
Tokumoto T
,
Kondo A
,
Miwa J
,
Horiguchi R
,
Tokumoto M
,
Nagahama Y
,
Okida N
,
Ishikawa K
.
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During Xenopus oocyte maturation, the amount of a 48 kDa protein detected in the 26S proteasome fraction (p48) decreased markedly during oocyte maturation to the low levels seen in unfertilized eggs. The results indicate that the interaction of at least one protein with the 26S proteasome changes during oocyte maturation and early development. An alteration in proteasome function may be important for the regulation of developmental events, such as the rapid cell cycle, in the early embryo. In this study, we identified p48. p48 was purified by conventional column chromatography. The resulting purified fraction contained two other proteins with molecular masses of 30 (p30) and 37 (p37) kDa. cDNAs encode elongation factor-1gamma and delta were obtained by an immuno-screening method using polyclonal antibodies against purified p48 complex, which recognized p48 and p37. N-terminal amino acid sequence analysis of p30 revealed that it was identical to EF-1beta. To identify the p48 complex bound to the 26S proteasome as EF-1betagammadelta, antibodies were raised against the components of purified p48 complex. Recombinant EF-1 beta,gamma and delta were expressed in Escherichia coli, and an antibody was raised against purified recombinant EF-1gamma. Cross-reactivity of the antibodies toward the p48 complex and recombinant proteins showed it to be specific for each component. These results indicate that the p48 complex bound to the 26S proteasome is the EF-1 complex. MPF phosphorylated EF-1gamma was shown to bind to the 26S proteasome. When EF-1gamma is phosphorylated by MPF, the association is stabilized. p48 bound to the 26S proteasome is identified as the EF-1gamma. EF-1 complex is associated with the 26S proteasome in Xenopus oocytes and the interaction is stabilized by MPF-mediated phosphorylation.
Figure 1. Purification of p48 The p48-containing fraction was purified as described in Materials and Methods. A, Q-Sepharose column chromatography: 35â80 % ammonium sulfate fraction was applied to a Q-Sepharose column (2.6 Ã 10 cm). Proteins were eluted with a linear gradient of NaCl and the 0.3â0.5 M eluate was collected in 10 ml fractions. B, SP-Sepharose column chromatography: p48-containing fractions from a Q-Sepharose column were pooled and applied to a SP-Sepharose column (1.6 Ã 10 cm). Proteins were eluted with a linear gradient of NaCl and the 0â0.25 M eluate was collected in 10 ml fractions. Fractions were assessed by immunoblotting using anti-Xenopus 20S proteasome. Protein bands of p48 and subunits of 20S proteasome are indicated by arrows and square brackets, respectively. Proteasome activity of the fractions toward a fluorogenic peptide substrate (Suc-Leu-Leu-Val-Tyr-MCA) was determined in the presence (â) and absence (â) of 0.05% SDS as described (21). Elution profile was monitored by absorbance at 280 nm (â).
Figure 2. Immunoblotting of purified p48 fraction The purified p48 fraction was analyzed by electrophoresis under denaturing conditions (12.0% gel) and either stained with Coomassie Brilliant Blue (CBBR) or immunostained with antibodies (α-20S: anti-Xenopus 20S proteasome, α-p48: anti-p48 fraction) after electroblotting. Protein bands are indicated by arrows. Molecular masses of standard proteins are indicated on the left.
Figure 3. Identification of p48 complex as an EF-1 complex Purified fractions were separated by electrophoresis under denaturing conditions (12.0% gel) and stained with Coomassie Brilliant Blue (CBBR) or immunostained with antibodies (α-p48: anti-p48 fraction, α-EF-1γ: anti-recombinant EF-1γ, α-p30: anti-p30 monoclonal antibody) after electroblotting. Lanes 1 to 5 were as follows; 1, purified p48 fraction; 2, purified 26S proteasome from immature oocytes; 3, recombinant EF-1β; 4, recombinant EF-1γ; 5, recombinant EF-1δ. Protein bands are indicated by arrows. Molecular masses of standard proteins are indicated on the left.
Figure 4. Alkaline phosphatase treatment of extracts from mature oocytes Extracts from mature oocytes were treated with calf intestinal alkaline phosphatase in the presence and absence of sodium vanadate (10 μM) as described in Methods. Lanes I and M correspond to extracts from immature and mature oocytes. Samples were immunoblotted with anti-proteasome antiserum (α-20S) and a guinea pig polyclonal antibody to recombinant EF-1γ (α-EF-1γ).
Figure 5. Phosphorylation of EF-1γ bound to the 26S proteasome (A) Phosphorylation of p48 complex and the 26S proteasome by MPF. Purified p48 complex and the 26S proteasome from immature oocytes were treated with or without MPF as indicated in the presence of [γ-32P]-ATP. After incubation at 30°C for 1 hour the reaction was stopped by addition of SDS-PAGE sample buffer. 32P-labeled proteins were resolved by SDS-PAGE followed by autoradiography on Imaging plates (Fuji Film). (B) Immunoprecipitation of p48 complex by anti-EF-1 complex. The 26S proteasome fractions were treated with affinity purified anti-EF-1 complex (Anti) or control IgG (Cont) and EF-1 components were immunoprecipitated as described previously [37]. Supernatants (S) and precipitates (P) were resolved by SDS-PAGE followed by autoradiography. Arrows indicate 32P-labeled 48, 37 and 30 kDa protein bands. Image analysis was performed using a Molecular Imager FX (BioRad).
Figure 6. Stabilization of association between EF-1 complex and the 26S proteasome by phosphorylation with MPF (A) The 26S proteasome fraction from Xenopus immature oocytes was resolved by electrophoresis under denaturing conditions (12 % gel) and immunostained with anti-goldfish 26S proteasome polyclonal antibody. (B) The 26S proteasome from immature oocytes was treated with MPF for 1 hour at 30°C. Samples were treated with affinity purified anti-goldfish 26S proteasome (Anti) or control IgG (Cont) and the 26S proteasomes were immunoprecipitated. Untreated sample (Total) and precipitates were resolved by SDS-PAGE followed by autoradiography. Arrows indicate 32P-labeled 48, 37 and 30 kDa protein bands. (C) The 26S proteasome from immature oocytes was treated with (MPF+) or without (MPF-) MPF for 1 hour at 30°C. Samples were immunoprecipitated using affinity-purified anti-goldfish 26S proteasome. Supernatants (S) and precipitates (P) were immunoblotted with antibodies (α-EF-1γ; anti-recombinant EF-1γ: α-GC4/5; anti-20S proteasome α2 subunit mouse monoclonal antibody). Protein bands of EF-1γ (p48) and α2 subunit of 20S proteasome (p25) are indicated by arrows. (D) Samples were analyzed by chromatography on a G4000SWXL column equilibrated with 50 mM Tris-HCl buffer, pH 7.5, containing 20% glycerol, 10 mM 2-mercaptoethanol and 0.1 mM ATP as described previously [36] in the presence of 0.5 M NaCl. Fractions were assessed by immunoblotting using a mixture of anti-Xenopus 20S proteasome and anti-EF-1γ polyclonal antibodies (α-20S+α-EF-1γ) or anti-Xenopus 20S proteasome (α-20S). Protein bands of EF-1γ (p48) and subunits of 20S proteasome are indicated by the arrow and square brackets, respectively.
Figure 7. Changes in association between EF-1 complex and the 26S proteasome during oocyte maturation (A) Immunoblotting of the purified 26S proteasomes. 26S proteasomes were analyzed by electrophoresis under denaturing conditions (12.0% gel) and either stained with Coomassie Brilliant Blue (CBBR) or immunostained with antibodies (α-GC4/5; anti-20S proteasome α2 subunit mouse monoclonal antibody, α-EF-1γ: anti-recombinant EF-1γ) after electroblotting. Lanes I and M indicate 26S proteasomes from immature and mature oocytes, respectively. (B) Oocyte extracts from Xenopus immature and mature oocytes were immunoprecipitated using affinity-purified anti-goldfish 26S proteasome. Precipitates were immunoblotted with antibodies. Lanes I and M indicate extract from immature and mature oocytes, respectively. Protein bands of EF-1γ (p48) and α2 subunit of 20S proteasome (p25) are indicated by arrows. Molecular masses of standard proteins are indicated at the left.
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