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OsHrd3 is necessary for maintaining the quality of endoplasmic reticulum-derived protein bodies in rice endosperm.

Ohta M, Takaiwa F - J. Exp. Bot. (2015)

Bottom Line: Co-immunoprecipitation experiments demonstrated that OsHrd3 interacts with components of the Hrd1 ubiquitin ligase complex such as OsOS-9 and OsHrd1 in rice protoplasts.Endosperm-specific suppression of OsHrd3 in transgenic rice reduced the levels of polyubiquitinated proteins and resulted in unfolded protein responses (UPRs) in the endosperm, suggesting that OsHrd3-mediated polyubiquitination plays an important role in ER quality control.Therefore, the quality of protein bodies is maintained by polyubiquitination of unfolded SSPs through the Hrd1 ubiquitin ligase system in rice endosperm.

View Article: PubMed Central - PubMed

Affiliation: Functional Transgenic Crops Research Unit, Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan.

No MeSH data available.


Related in: MedlinePlus

OsHrd3 is required for polyubiquitination of unfolded proteins in rice endosperm. (A) The construct used for OsHrd3 knockdown (OsHrd3 KD); 35S P, Cauliflower mosaic virus 35S promoter (AF485783); HPT, hygromycin phosphotransferase coding region (K01193); Ag7 T, gene 7 terminator (AF85783); 16 kD P, promoter region of the gene encoding 16kDa prolamin (AY427574); RAPint, an intron from the rice aspartic protease gene (D32165); 16 kD T, 16kDa prolamin terminator. (B and C) Levels of polyubiquitinated proteins are reduced in OsHrd3 KD seeds. Seeds (14 DAF) from wild-type (WT) and OsHrd3 KD plants were dehulled and treated with either 0.1% dimethylsulphoxide (–) or 100 μM MG132 (+) for 24h and then treated with 20 μM PR-619 for 1h. Then, total proteins were extracted from the seeds with SDS–urea buffer containing 2-mercaptoethanol. The total proteins were separated by SDS–PAGE, followed by immunoblot analyses using an antibody against ubiquitin–protein conjugates (B) or Coomassie Brilliant Blue staining (C).
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Figure 2: OsHrd3 is required for polyubiquitination of unfolded proteins in rice endosperm. (A) The construct used for OsHrd3 knockdown (OsHrd3 KD); 35S P, Cauliflower mosaic virus 35S promoter (AF485783); HPT, hygromycin phosphotransferase coding region (K01193); Ag7 T, gene 7 terminator (AF85783); 16 kD P, promoter region of the gene encoding 16kDa prolamin (AY427574); RAPint, an intron from the rice aspartic protease gene (D32165); 16 kD T, 16kDa prolamin terminator. (B and C) Levels of polyubiquitinated proteins are reduced in OsHrd3 KD seeds. Seeds (14 DAF) from wild-type (WT) and OsHrd3 KD plants were dehulled and treated with either 0.1% dimethylsulphoxide (–) or 100 μM MG132 (+) for 24h and then treated with 20 μM PR-619 for 1h. Then, total proteins were extracted from the seeds with SDS–urea buffer containing 2-mercaptoethanol. The total proteins were separated by SDS–PAGE, followed by immunoblot analyses using an antibody against ubiquitin–protein conjugates (B) or Coomassie Brilliant Blue staining (C).

Mentions: Maturing rice seeds produce a large amount of SSPs. Thus, a significant amount of unfolded protein is likely to be produced by stochastic errors during protein synthesis, perturbation by adverse environmental changes, and an imbalance in stoichiometry among components of the protein bodies. However, it is unclear how the ER in developing seeds discriminates and removes such unfolded proteins. To elucidate the role of a quality control system in rice seeds, transgenic rice plants were generated with suppressed expression of OsHrd3 in the endosperm under the control of the 16kDa prolamin (Os03g0766200) promoter (Fig. 2A). Since mRNAs for OsHrd3 and 16kDa prolamin were detected at 7DAF (Fig. 3B; Supplementary Fig. S2D at JXB online), the 16kDa prolamin promoter is suitable for suppression of OsHrd3 expression. RT-PCR analysis showed that the level of OsHrd3 transcript was lower in OsHrd3 KD seeds than in WT seeds (Fig. 3B).


OsHrd3 is necessary for maintaining the quality of endoplasmic reticulum-derived protein bodies in rice endosperm.

Ohta M, Takaiwa F - J. Exp. Bot. (2015)

OsHrd3 is required for polyubiquitination of unfolded proteins in rice endosperm. (A) The construct used for OsHrd3 knockdown (OsHrd3 KD); 35S P, Cauliflower mosaic virus 35S promoter (AF485783); HPT, hygromycin phosphotransferase coding region (K01193); Ag7 T, gene 7 terminator (AF85783); 16 kD P, promoter region of the gene encoding 16kDa prolamin (AY427574); RAPint, an intron from the rice aspartic protease gene (D32165); 16 kD T, 16kDa prolamin terminator. (B and C) Levels of polyubiquitinated proteins are reduced in OsHrd3 KD seeds. Seeds (14 DAF) from wild-type (WT) and OsHrd3 KD plants were dehulled and treated with either 0.1% dimethylsulphoxide (–) or 100 μM MG132 (+) for 24h and then treated with 20 μM PR-619 for 1h. Then, total proteins were extracted from the seeds with SDS–urea buffer containing 2-mercaptoethanol. The total proteins were separated by SDS–PAGE, followed by immunoblot analyses using an antibody against ubiquitin–protein conjugates (B) or Coomassie Brilliant Blue staining (C).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4507767&req=5

Figure 2: OsHrd3 is required for polyubiquitination of unfolded proteins in rice endosperm. (A) The construct used for OsHrd3 knockdown (OsHrd3 KD); 35S P, Cauliflower mosaic virus 35S promoter (AF485783); HPT, hygromycin phosphotransferase coding region (K01193); Ag7 T, gene 7 terminator (AF85783); 16 kD P, promoter region of the gene encoding 16kDa prolamin (AY427574); RAPint, an intron from the rice aspartic protease gene (D32165); 16 kD T, 16kDa prolamin terminator. (B and C) Levels of polyubiquitinated proteins are reduced in OsHrd3 KD seeds. Seeds (14 DAF) from wild-type (WT) and OsHrd3 KD plants were dehulled and treated with either 0.1% dimethylsulphoxide (–) or 100 μM MG132 (+) for 24h and then treated with 20 μM PR-619 for 1h. Then, total proteins were extracted from the seeds with SDS–urea buffer containing 2-mercaptoethanol. The total proteins were separated by SDS–PAGE, followed by immunoblot analyses using an antibody against ubiquitin–protein conjugates (B) or Coomassie Brilliant Blue staining (C).
Mentions: Maturing rice seeds produce a large amount of SSPs. Thus, a significant amount of unfolded protein is likely to be produced by stochastic errors during protein synthesis, perturbation by adverse environmental changes, and an imbalance in stoichiometry among components of the protein bodies. However, it is unclear how the ER in developing seeds discriminates and removes such unfolded proteins. To elucidate the role of a quality control system in rice seeds, transgenic rice plants were generated with suppressed expression of OsHrd3 in the endosperm under the control of the 16kDa prolamin (Os03g0766200) promoter (Fig. 2A). Since mRNAs for OsHrd3 and 16kDa prolamin were detected at 7DAF (Fig. 3B; Supplementary Fig. S2D at JXB online), the 16kDa prolamin promoter is suitable for suppression of OsHrd3 expression. RT-PCR analysis showed that the level of OsHrd3 transcript was lower in OsHrd3 KD seeds than in WT seeds (Fig. 3B).

Bottom Line: Co-immunoprecipitation experiments demonstrated that OsHrd3 interacts with components of the Hrd1 ubiquitin ligase complex such as OsOS-9 and OsHrd1 in rice protoplasts.Endosperm-specific suppression of OsHrd3 in transgenic rice reduced the levels of polyubiquitinated proteins and resulted in unfolded protein responses (UPRs) in the endosperm, suggesting that OsHrd3-mediated polyubiquitination plays an important role in ER quality control.Therefore, the quality of protein bodies is maintained by polyubiquitination of unfolded SSPs through the Hrd1 ubiquitin ligase system in rice endosperm.

View Article: PubMed Central - PubMed

Affiliation: Functional Transgenic Crops Research Unit, Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan.

No MeSH data available.


Related in: MedlinePlus