1. The Arabidopsis Homolog of the Mammalian OS-9 Protein Plays a Key Role in the Endoplasmic Reticulum-Associated Degradation of Misfolded Receptor-Like Kinases 
Wei Su, Yidan Liu, Yang Xia, Zhi Hong, Jianming Li 
Molecular Plant 
Volume 5, Issue 4, Pages (July 2012)
DOI: /mp/sss042
Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

2. Figure 1
AtOS9 Is a Likely Homolog of Yos9/OS-9 and Localizes in the ER.
(A) Alignment of the MRH domains between Yos9/OS-9 and their plant homologs. Yos9 (accession number: NP_010342), human Os-9 (NP_006803), and predicted Yos9/OS-9 homologs from Arabidopsis (AtOS9, NP_568525), Physcomitrella patens (PpOS9, XP_ ), Oryza sativa (OsOS9, NP_ ), Zea mays (ZmOS9, NP_ ), and mouse (MmOS9, NP_ ) were aligned using the ClustalW program. The aligned amino acid sequences were color shaded by the Boxshade program at the Mobyle portal ( The regions containing the predicted MRH domains are shown here, while the alignment of full-length proteins is presented in Supplemental Figure 1. Identical residues are colored in red while similar residues are shaded in cyan. Stars indicated amino acids directly involved in N-glycan binding (Satoh et al., 2010).
(B, D) Immunoblot analysis of AtOS9. Equal amounts of total proteins extracted from 2-week-old seedlings treated with or without 5 μg ml−1 TM were treated without or with EndoH, separated by 10% SDS–PAGE, and analyzed by immunoblot using an anti-AtOS9 antibody. Coomassie blue staining of the small subunit of the Arabidopsis Rubisco (RbcS) serves as a loading control.
(C) Confocal microscopic analysis of AtOS9. Shown from left to right are fluorescence patterns of the AtOS9–GFP fusion protein (green), the ER-localized HDEL-tagged RFP (red), and the superimposed image of the green and red fluorescent signals in Agrobacterium-infiltrated tobacco leaf epidermal cells.
Molecular Plant 2012 5, DOI: ( /mp/sss042)
Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

3. Figure 2
Identification of a T-DNA Insertional atos9-t Mutant Hypersensitive to TM.
(A) Schematic presentation of the AtOS9 gene structure. The thin lines represent introns, black bars denote amino acid-encoding exons, and white bars indicate untranslated regions of the first and last exons. The position and orientation of the inserted T-DNA are indicated.
(B) Immunoblot analysis of AtOS9. Equal amounts of total proteins extracted from seedlings of wild-type, atos9-t, and bri1-9 were separated by 10% SDS–PAGE and analyzed by immunoblot using an anti-AtOS9 antibody. Asterisk indicates a non-specific band for loading control.
(C) Images of 4-week-old soil-grown seedlings of wild-type and atos9-t.
(D) Images of 4-week-old seedlings of wild-type and atos9-t mutant grown on TM-containing ½ MS medium.
Molecular Plant 2012 5, DOI: ( /mp/sss042)
Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

4. Figure 3
The atos9-t Mutation Inhibits the ERAD of bri1-9 and Suppresses the bri1-9 Dwarfism.
(A) Immunoblot analysis of the BRI1/bri1-9 abundance. Equal amounts of total proteins extracted from 2-week-old seedlings were treated with or without Endo H, separated by 7% SDS–PAGE, and analyzed by immunoblot with anti-BRI1 antibody.
(B) Immunoblot analysis of the bri1-9 stability. Two-week-old seedlings were transferred into liquid ½ MS medium containing 180 μM CHX. Equal amounts of seedlings were removed at indicated incubation times to extract total proteins in 2×SDS sample buffer, which were subsequently analyzed by immunoblot with the anti-BRI1 antibody.
(C–E) Images of 4-week-old soil-grown plants (C), 5-day-old dark-grown seedlings (D), and 2-month-old mature plants (E) of wild-type, bri1-9, and atos9-t bri1-9.
(F) The root-growth inhibition assay. Root lengths of 7-day-old seedlings grown on BL-containing medium were measured and presented as the relative value of the average root length of BL-treated seedlings to that of untreated seedlings of the same genotype. Each data point represents the average of ∼40 seedlings of duplicated experiments. Error bars denote standard error.
(G) Immunoblot analysis of the BES1 phosphorylation status. Equal amounts of total proteins extracted from 2-week-old seedlings treated with or without 1 μM BL for 1 h were separated by 10% SDS–PAGE and analyzed by immunoblot using an anti-BES1 antiserum. In (A), (B), and (G), coomassie blue staining of RbcS serves as a loading control.
Molecular Plant 2012 5, DOI: ( /mp/sss042)
Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

5. Figure 4
The atos9-t Mutation Blocks the ERAD of the Mutant BR Receptor bri1-9.
(A) Phenotypic comparison between bri1-9, atos9-t bri1-9, and three representative gAtOS9–GFP atos9-t bri1-9 transgenic lines expressing a genomic AtOS9–GFP transgene.
(B, C) Immunoblot analysis of AtOS9/AtOS9–GFP (B) and bri1-9 (C). Equal amounts of total proteins extracted from 2-week-old seedlings were separated by 10% SDS–PAGE and analyzed by an anti-AtOS9 antibody (B) or by an anti-BRI1 antibody (C).
(D, E) Images of 4-week-old (D) and 2-month-old (E) soil-grown plants of wild-type, bri1-9, and ebs6-1 bri1-9.
(F) Immunoblot analysis of the BRI1/bri1-9 abundance. Equal amounts of total proteins extracted from 2-week-old seedlings were treated with or without Endo H, separated by 7% SDS–PAGE, and analyzed by immunoblot with anti-BRI1 antibody. In (B), (C), and (F), coomassie blue staining of RbcS serves as a loading control.
Molecular Plant 2012 5, DOI: ( /mp/sss042)
Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

6. Figure 5
The atos9-t Mutation Inhibits the ERAD of bri1-5 and Misfolded EFR.
(A) Immunoblot analysis of bri1-5. Total proteins extracted from 2-week-old seedlings were treated with or without Endo H, separated by 7% SDS–PAGE, and analyzed by immunoblot with anti-BRI1 antibody.
(B–D) Images of 4-week-old soil-grown plants (B), 5-day-old dark-grown seedlings (C), and 2-month-old mature plants (D) of wild-type, bri1-5, and atos9-t bri1-5.
(E) Immunoblot analysis of EFR. Total proteins extracted from 2-week-old seedlings were treated with or without Endo H, separated by 7% SDS–PAGE, and analyzed by immunoblot with an anti-EFR antibody. In both (A) and (E), coomassie blue staining of RbcS serves as a loading control.
Molecular Plant 2012 5, DOI: ( /mp/sss042)
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7. Figure 6 The MRH Domain Is Important for the AtOS9 Function.
Shown here are images of representative transgenic atos9-t bri1-9 lines containing an empty vector or expressing the wild-type or a mutant p35S:AtOS9 transgene carrying one of three indicated single amino acid changes.
Molecular Plant 2012 5, DOI: ( /mp/sss042)
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8. Figure 7 AtOS9 Interacts Biochemically and Genetically with EBS5.
(A) An in vitro pull-down assay. Equal amounts of the MBP–EBS5 fusion protein still bound to amylose resin were incubated with GST–AtOS9 or GST. After extensive washing, the proteins remained on the resin were dissolved in 2×SDS sample buffer, separated by 10% SDS–PAGE, and analyzed by immunoblot with an anti-GST antibody. Commassie blue staining of MBP–EBS5 shows the amounts of MBP–EBS5 used in the binding assay.
(B) Immunoblot analysis of AtOS9. Equal amounts of total proteins extracted from 2-week-old seedlings were separated by 10% SDS–PAGE and analyzed by immunoblot with an anti-AtOS9 antibody. Star indicates a cross-reacting band used for the loading control.
(C) Immunoblot analysis of the AtOS9 stability. Two-week-old seedlings were transferred into liquid ½ MS medium containing 180 μM CHX. Equal amounts of seedlings were removed at indicated incubation times to extract total proteins in 2×SDS sample buffer, which were subsequently analyzed by immunoblot with the anti-AtOS9 antibody. Coomassie blue staining of RbcS was used for a loading control.
(D) Shown here (from left to right) are images of 4-week-old soil-grown plants of wild-type, atos9-t ebs5-3 bri1-9, bri1-9, ebs5-3 bri1-9, and atos9-t bri1-9.
Molecular Plant 2012 5, DOI: ( /mp/sss042)
Copyright © 2012 The Authors. All rights reserved. Terms and Conditions