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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.

Show MeSH
Binary vector constructs. Ag 7, Agrobacterium gene 7 terminator; HPT, hygromycin phosphotransferase coding region; 35S pro, CaMV 35S promoter; 2.3 K GluB1 pro, 2.3 kb rice glutelin B1 promoter; GluA2, wild-type rice glutelin A2 coding region; GluB ter, 0.65 kb glutelin B1 terminator; mGluA2, mutated-glutelin A2 coding region.
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fig1: Binary vector constructs. Ag 7, Agrobacterium gene 7 terminator; HPT, hygromycin phosphotransferase coding region; 35S pro, CaMV 35S promoter; 2.3 K GluB1 pro, 2.3 kb rice glutelin B1 promoter; GluA2, wild-type rice glutelin A2 coding region; GluB ter, 0.65 kb glutelin B1 terminator; mGluA2, mutated-glutelin A2 coding region.

Mentions: Gene cassettes consisted of the endosperm-specific 2.3 kb glutelin B1 (GluB1) promoter with 5′ UTR (Accession No. AY427569), the coding region of the glutelin A2 (GluA2) gene (Accession No. X05664), or mutated-GluA2 (mGluA2), in which the processing site (Asn-Gly) was replaced by Ala-Ala, and 0.65 kb GluB1 terminator (Accession No. X54314) were introduced into the multiple cloning sites of modified-pBluescript KS+ containing the Gateway recombination sites att L1 and att L2. Then, two gene cassettes were transferred from entry clones to a destination-binary vector (p35:HPT Ag7-GW; Wakasa et al., 2006) by LR clonase II enzyme Mix (Invitrogen, CA, USA). The binary vector constructs are shown in Fig. 1.


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

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

Binary vector constructs. Ag 7, Agrobacterium gene 7 terminator; HPT, hygromycin phosphotransferase coding region; 35S pro, CaMV 35S promoter; 2.3 K GluB1 pro, 2.3 kb rice glutelin B1 promoter; GluA2, wild-type rice glutelin A2 coding region; GluB ter, 0.65 kb glutelin B1 terminator; mGluA2, mutated-glutelin A2 coding region.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Binary vector constructs. Ag 7, Agrobacterium gene 7 terminator; HPT, hygromycin phosphotransferase coding region; 35S pro, CaMV 35S promoter; 2.3 K GluB1 pro, 2.3 kb rice glutelin B1 promoter; GluA2, wild-type rice glutelin A2 coding region; GluB ter, 0.65 kb glutelin B1 terminator; mGluA2, mutated-glutelin A2 coding region.
Mentions: Gene cassettes consisted of the endosperm-specific 2.3 kb glutelin B1 (GluB1) promoter with 5′ UTR (Accession No. AY427569), the coding region of the glutelin A2 (GluA2) gene (Accession No. X05664), or mutated-GluA2 (mGluA2), in which the processing site (Asn-Gly) was replaced by Ala-Ala, and 0.65 kb GluB1 terminator (Accession No. X54314) were introduced into the multiple cloning sites of modified-pBluescript KS+ containing the Gateway recombination sites att L1 and att L2. Then, two gene cassettes were transferred from entry clones to a destination-binary vector (p35:HPT Ag7-GW; Wakasa et al., 2006) by LR clonase II enzyme Mix (Invitrogen, CA, USA). The binary vector constructs are shown in Fig. 1.

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