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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: However, it was previously unclear how such unfolded proteins are selected among synthesized products and removed from the ER to maintain protein quality in the endosperm.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.

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

Interaction between OsHrd3 and components of the Hrd1 ubiquitin ligase complex. Immunoprecipitation of OsHrd3-FLAGs. Protein extracts from rice protoplasts expressing OsHrd1-HA, OsOS-9-HA, and OsHrd3-FLAG were subjected to immunoprecipitation using anti-FLAG M2 magnetic beads. The immunoprecipitates were analysed by immunoblot analysis using anti-FLAG–horseradish peroxidase (HRP) and anti-HA–HRP conjugated antibodies. C represents 2% (v/v) of the starting crude lysate used for immunoprecipitation. P represents immunoprecipitated proteins.
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Figure 1: Interaction between OsHrd3 and components of the Hrd1 ubiquitin ligase complex. Immunoprecipitation of OsHrd3-FLAGs. Protein extracts from rice protoplasts expressing OsHrd1-HA, OsOS-9-HA, and OsHrd3-FLAG were subjected to immunoprecipitation using anti-FLAG M2 magnetic beads. The immunoprecipitates were analysed by immunoblot analysis using anti-FLAG–horseradish peroxidase (HRP) and anti-HA–HRP conjugated antibodies. C represents 2% (v/v) of the starting crude lysate used for immunoprecipitation. P represents immunoprecipitated proteins.

Mentions: Hrd3/SEL1L is a component of the Hrd1 ubiquitin ligase complex. In yeast, the Hrd1 complex consists of Hrd3p and Der1p, including the ER lectin Yos9p bound to Hrd3p (Carvalho et al., 2006; Denic et al., 2006). Rice genes encoding homologues of the yeast Hrd1 and Yos9p were found in the database (RAP-DB, http://rapdb.dna.affrc.go.jp/). To examine the possibility that OsHrd3 also forms a complex with these proteins, the interaction between OsHrd3 and both OsHrd1 (Os06g0301000) and OsOS-9 (Os06g0644800), which are putative homologues of yeast Hrd1 and Yos-9p, respectively, was investigated. OsHrd1 and OsOS-9 were fused with 2× HA tag sequence at their C-termini. Plasmid DNA harboring OsHrd3-FLAG, together with OsHrd1-HA or OsOS-9-HA, was then transfected into rice protoplasts and Co-IP analysis was performed with an antibody against FLAG tag (Ohta et al., 2013). As shown in Fig. 1, OsHrd1-HA and OsOS-9-HA co-precipitated with OsHrd3-FLAG, demonstrating that OsHrd1 and OsOS-9 can interact with OsHrd3. These data suggest that OsHrd3 could form a complex with OsHrd1 and OsOS-9.


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

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

Interaction between OsHrd3 and components of the Hrd1 ubiquitin ligase complex. Immunoprecipitation of OsHrd3-FLAGs. Protein extracts from rice protoplasts expressing OsHrd1-HA, OsOS-9-HA, and OsHrd3-FLAG were subjected to immunoprecipitation using anti-FLAG M2 magnetic beads. The immunoprecipitates were analysed by immunoblot analysis using anti-FLAG–horseradish peroxidase (HRP) and anti-HA–HRP conjugated antibodies. C represents 2% (v/v) of the starting crude lysate used for immunoprecipitation. P represents immunoprecipitated proteins.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4507767&req=5

Figure 1: Interaction between OsHrd3 and components of the Hrd1 ubiquitin ligase complex. Immunoprecipitation of OsHrd3-FLAGs. Protein extracts from rice protoplasts expressing OsHrd1-HA, OsOS-9-HA, and OsHrd3-FLAG were subjected to immunoprecipitation using anti-FLAG M2 magnetic beads. The immunoprecipitates were analysed by immunoblot analysis using anti-FLAG–horseradish peroxidase (HRP) and anti-HA–HRP conjugated antibodies. C represents 2% (v/v) of the starting crude lysate used for immunoprecipitation. P represents immunoprecipitated proteins.
Mentions: Hrd3/SEL1L is a component of the Hrd1 ubiquitin ligase complex. In yeast, the Hrd1 complex consists of Hrd3p and Der1p, including the ER lectin Yos9p bound to Hrd3p (Carvalho et al., 2006; Denic et al., 2006). Rice genes encoding homologues of the yeast Hrd1 and Yos9p were found in the database (RAP-DB, http://rapdb.dna.affrc.go.jp/). To examine the possibility that OsHrd3 also forms a complex with these proteins, the interaction between OsHrd3 and both OsHrd1 (Os06g0301000) and OsOS-9 (Os06g0644800), which are putative homologues of yeast Hrd1 and Yos-9p, respectively, was investigated. OsHrd1 and OsOS-9 were fused with 2× HA tag sequence at their C-termini. Plasmid DNA harboring OsHrd3-FLAG, together with OsHrd1-HA or OsOS-9-HA, was then transfected into rice protoplasts and Co-IP analysis was performed with an antibody against FLAG tag (Ohta et al., 2013). As shown in Fig. 1, OsHrd1-HA and OsOS-9-HA co-precipitated with OsHrd3-FLAG, demonstrating that OsHrd1 and OsOS-9 can interact with OsHrd3. These data suggest that OsHrd3 could form a complex with OsHrd1 and OsOS-9.

Bottom Line: However, it was previously unclear how such unfolded proteins are selected among synthesized products and removed from the ER to maintain protein quality in the endosperm.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.

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