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Expression of unprocessed glutelin precursor alters polymerization without affecting trafficking and accumulation.

Wakasa Y, Yang L, Hirose S, Takaiwa F - J. Exp. Bot. (2009)

Bottom Line: In order to investigate the functional role of this processing and its effect on folding assembly, wild-type GluA2 and its mutant cDNA (mGluA2), in which the conserved processing site (Asn-Gly) at the junction between the acidic and basic chains was replaced with Ala-Ala, were expressed under the control of the endosperm-specific GluB1 promoter in the mutant rice a123 line lacking glutelin GluA1, GluA2, and GluB4.Furthermore, the mGluA2 precursor in the glutelin fraction was deposited in PB-II by forming a quite different complex from the processed mature GluA2 products.These results indicate that post-translational processing of glutelin is not necessary for trafficking and stable accumulation in PB-II, but is required for the formation of the higher-order structure required for stacking in PB-II.

View Article: PubMed Central - PubMed

Affiliation: Transgenic Crop Research and Development Center, National Institute of Agrobiological Sciences, Kannondai 3-1-3, Tsukuba, Ibaraki 305-8604, Japan.

ABSTRACT
Rice glutelin is synthesized as a precursor in the endosperm endoplasmic reticulum and then deposited within the protein storage vacuole protein body-II (PB-II) as an aggregate, with a high degree of polymerized higher-order structure comprising mature acidic and basic subunits after post-translation processing cleavage. In order to investigate the functional role of this processing and its effect on folding assembly, wild-type GluA2 and its mutant cDNA (mGluA2), in which the conserved processing site (Asn-Gly) at the junction between the acidic and basic chains was replaced with Ala-Ala, were expressed under the control of the endosperm-specific GluB1 promoter in the mutant rice a123 line lacking glutelin GluA1, GluA2, and GluB4. The mGluA2 precursor was synthesized and stably targeted to PB-II without processing in the transgenic rice seeds like the wild-type GluA2. Notably, the saline-soluble mGluA2 precursor assembled with the other type of processed glutelin GluB as a trimer in PB-II, although such hetero-assembly with GluB was not detected in the transformant containing the processed GluA. Furthermore, the mGluA2 precursor in the glutelin fraction was deposited in PB-II by forming a quite different complex from the processed mature GluA2 products. These results indicate that post-translational processing of glutelin is not necessary for trafficking and stable accumulation in PB-II, but is required for the formation of the higher-order structure required for stacking in PB-II.

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SDS-PAGE and immuno-blot analysis of transgene products extracted by total protein extraction buffer after pre-extraction by globulin extraction buffer with or without 5% 2-mecaptoethanol. The globulin extraction buffer-extracted fraction (supernatant) was 5-fold concentrated by acetone precipitation compared with the pellet fraction. (A) Each extracted protein fraction was subjected to SDS-PAGE and immuno-blot analysis using anti-GluA and anti-GluB antibodies. (B) In the globulin fraction, in order to detect the matured GluB signal, overexposed X-ray film is also shown.
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fig6: SDS-PAGE and immuno-blot analysis of transgene products extracted by total protein extraction buffer after pre-extraction by globulin extraction buffer with or without 5% 2-mecaptoethanol. The globulin extraction buffer-extracted fraction (supernatant) was 5-fold concentrated by acetone precipitation compared with the pellet fraction. (A) Each extracted protein fraction was subjected to SDS-PAGE and immuno-blot analysis using anti-GluA and anti-GluB antibodies. (B) In the globulin fraction, in order to detect the matured GluB signal, overexposed X-ray film is also shown.

Mentions: As shown in Fig. 6, the GluA2 or mGluA2 product was examined by extraction with the saline solution in the presence or absence of 2-MER. Addition of 2-MER apparently enhanced the extraction efficiency of the GluA2 and mGluA2 products in the globulin fraction, resulting in a yield of 12.2% and 22.8% of total seed protein, respectively (Table 1). This is in contrast with the extraction efficiency of 2.5% and 9.6% in the absence of 2-MER. This evidence indicates that disulphide bonds are involved in the interaction between GluA and other glutelins. Notably, the GluA2 precursor could be easily extracted with saline solution, irrespective of 2-MER, whereas a reducing agent was required for the extraction of mature GluB subunits (Fig. 6A). These results indicate that interactions among GluA and GluB products are mainly mediated by disulphide bonds.


Expression of unprocessed glutelin precursor alters polymerization without affecting trafficking and accumulation.

Wakasa Y, Yang L, Hirose S, Takaiwa F - J. Exp. Bot. (2009)

SDS-PAGE and immuno-blot analysis of transgene products extracted by total protein extraction buffer after pre-extraction by globulin extraction buffer with or without 5% 2-mecaptoethanol. The globulin extraction buffer-extracted fraction (supernatant) was 5-fold concentrated by acetone precipitation compared with the pellet fraction. (A) Each extracted protein fraction was subjected to SDS-PAGE and immuno-blot analysis using anti-GluA and anti-GluB antibodies. (B) In the globulin fraction, in order to detect the matured GluB signal, overexposed X-ray film is also shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2724699&req=5

fig6: SDS-PAGE and immuno-blot analysis of transgene products extracted by total protein extraction buffer after pre-extraction by globulin extraction buffer with or without 5% 2-mecaptoethanol. The globulin extraction buffer-extracted fraction (supernatant) was 5-fold concentrated by acetone precipitation compared with the pellet fraction. (A) Each extracted protein fraction was subjected to SDS-PAGE and immuno-blot analysis using anti-GluA and anti-GluB antibodies. (B) In the globulin fraction, in order to detect the matured GluB signal, overexposed X-ray film is also shown.
Mentions: As shown in Fig. 6, the GluA2 or mGluA2 product was examined by extraction with the saline solution in the presence or absence of 2-MER. Addition of 2-MER apparently enhanced the extraction efficiency of the GluA2 and mGluA2 products in the globulin fraction, resulting in a yield of 12.2% and 22.8% of total seed protein, respectively (Table 1). This is in contrast with the extraction efficiency of 2.5% and 9.6% in the absence of 2-MER. This evidence indicates that disulphide bonds are involved in the interaction between GluA and other glutelins. Notably, the GluA2 precursor could be easily extracted with saline solution, irrespective of 2-MER, whereas a reducing agent was required for the extraction of mature GluB subunits (Fig. 6A). These results indicate that interactions among GluA and GluB products are mainly mediated by disulphide bonds.

Bottom Line: In order to investigate the functional role of this processing and its effect on folding assembly, wild-type GluA2 and its mutant cDNA (mGluA2), in which the conserved processing site (Asn-Gly) at the junction between the acidic and basic chains was replaced with Ala-Ala, were expressed under the control of the endosperm-specific GluB1 promoter in the mutant rice a123 line lacking glutelin GluA1, GluA2, and GluB4.Furthermore, the mGluA2 precursor in the glutelin fraction was deposited in PB-II by forming a quite different complex from the processed mature GluA2 products.These results indicate that post-translational processing of glutelin is not necessary for trafficking and stable accumulation in PB-II, but is required for the formation of the higher-order structure required for stacking in PB-II.

View Article: PubMed Central - PubMed

Affiliation: Transgenic Crop Research and Development Center, National Institute of Agrobiological Sciences, Kannondai 3-1-3, Tsukuba, Ibaraki 305-8604, Japan.

ABSTRACT
Rice glutelin is synthesized as a precursor in the endosperm endoplasmic reticulum and then deposited within the protein storage vacuole protein body-II (PB-II) as an aggregate, with a high degree of polymerized higher-order structure comprising mature acidic and basic subunits after post-translation processing cleavage. In order to investigate the functional role of this processing and its effect on folding assembly, wild-type GluA2 and its mutant cDNA (mGluA2), in which the conserved processing site (Asn-Gly) at the junction between the acidic and basic chains was replaced with Ala-Ala, were expressed under the control of the endosperm-specific GluB1 promoter in the mutant rice a123 line lacking glutelin GluA1, GluA2, and GluB4. The mGluA2 precursor was synthesized and stably targeted to PB-II without processing in the transgenic rice seeds like the wild-type GluA2. Notably, the saline-soluble mGluA2 precursor assembled with the other type of processed glutelin GluB as a trimer in PB-II, although such hetero-assembly with GluB was not detected in the transformant containing the processed GluA. Furthermore, the mGluA2 precursor in the glutelin fraction was deposited in PB-II by forming a quite different complex from the processed mature GluA2 products. These results indicate that post-translational processing of glutelin is not necessary for trafficking and stable accumulation in PB-II, but is required for the formation of the higher-order structure required for stacking in PB-II.

Show MeSH