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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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Sequential extraction of seed proteins from Koshihikari, a123, and transgenic rice with GluA2 and mGluA2. (A) Each extracted protein fraction was subjected to SDS-PAGE and immuno-blot analysis using anti-GluA and anti-GluB antibody. (B) In the globulin fraction, overexposed X-ray film is also shown.
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fig5: Sequential extraction of seed proteins from Koshihikari, a123, and transgenic rice with GluA2 and mGluA2. (A) Each extracted protein fraction was subjected to SDS-PAGE and immuno-blot analysis using anti-GluA and anti-GluB antibody. (B) In the globulin fraction, overexposed X-ray film is also shown.

Mentions: As shown in Fig. 5, most of the GluA2 or mGluA2 products expressed in seeds of the a123 line could be extracted in the glutelin and residual prolamin fractions. It is interesting to note that a small portion of the mGluA2 product could be extracted in the globulin fraction, which was accompanied by a higher level of endogenous GluB product in the globulin fraction compared with that in the control a123 line. Since the GluB could not be detected in a globulin fraction from Koshihikari (Fig. 5B), recovery in the soluble fraction of the GluB product may be related to high expression of the mGluA2 precursor in mGluA2 transformant or low levels of mature GluA product in a123. By contrast, when GluA products are mainly accumulated as mature subunits in GluA transformant or Koshihikari, the GluB acidic subunits could not be extracted in the globulin fraction. These results suggest that the GluB subunits interact with the GluA precursor in a different manner from disulphide bonds and recovered as hetero-complexes in the saline solution.


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

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

Sequential extraction of seed proteins from Koshihikari, a123, and transgenic rice with GluA2 and mGluA2. (A) Each extracted protein fraction was subjected to SDS-PAGE and immuno-blot analysis using anti-GluA and anti-GluB antibody. (B) In the globulin fraction, 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

fig5: Sequential extraction of seed proteins from Koshihikari, a123, and transgenic rice with GluA2 and mGluA2. (A) Each extracted protein fraction was subjected to SDS-PAGE and immuno-blot analysis using anti-GluA and anti-GluB antibody. (B) In the globulin fraction, overexposed X-ray film is also shown.
Mentions: As shown in Fig. 5, most of the GluA2 or mGluA2 products expressed in seeds of the a123 line could be extracted in the glutelin and residual prolamin fractions. It is interesting to note that a small portion of the mGluA2 product could be extracted in the globulin fraction, which was accompanied by a higher level of endogenous GluB product in the globulin fraction compared with that in the control a123 line. Since the GluB could not be detected in a globulin fraction from Koshihikari (Fig. 5B), recovery in the soluble fraction of the GluB product may be related to high expression of the mGluA2 precursor in mGluA2 transformant or low levels of mature GluA product in a123. By contrast, when GluA products are mainly accumulated as mature subunits in GluA transformant or Koshihikari, the GluB acidic subunits could not be extracted in the globulin fraction. These results suggest that the GluB subunits interact with the GluA precursor in a different manner from disulphide bonds and recovered as hetero-complexes in the saline solution.

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