Limits...
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

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

License 1 - License 2
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.


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

License 1 - License 2
Show All Figures
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: 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