1. ERAD Tuning of the HRD1 Complex Component AtOS9 Is Modulated by an ER-Bound E2, UBC32 
Qian Chen, Ruijun Liu, Qian Wang, Qi Xie 
Molecular Plant 
Volume 10, Issue 6, Pages (June 2017)
DOI: /j.molp
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2. Figure 1
UBC32 Negatively Regulates AtOS9 at Both the Biochemical Level and the Genetic Level.
(A) UBC32 interacts with AtOS9 by co-IP assay. UBC32-GFP and AtOS9-myc were expressed in N. benthamiana leaf, and co-IP with GFP antibodies was performed. GFP-Trap_MA and GFP vector protein worked as negative controls. Black arrow indicates AtOS9-myc. Gray and blue arrows indicate UBC32-GFP and GFP, respectively. Asterisk indicates the modified AtOS9-myc.
(B) AtOS9 binds to UBC32 in planta by LCI. The pseudocolor bar shows the range of luminescence intensity in the image.
(C) AtOS9 was accumulated in ubc32 mutants compared with WT. Total proteins were extracted from WT, ubc32 and hrd3a mutants and subjected to SDS–PAGE. The internal AtOS9 level was detected by AtOS9 antibody. Ponceau S was used as loading control.
(D) The ubiquitination of AtOS9 is K48 linked but not K63 linked. Myc-GFP was used as the vector control. Myc-GFP and AtOS9-myc were immunoprecipitated by Myc antibody, and the ubiquitination form was then detected by K48 or K63 specific antibody. The line on the right of the immunoblot detected by K48 antibody indicates the position of the ubiquitinated AtOS9 protein.
(E) UBC32 promotes the degradation of AtOS9 in vivo. GFP was the vector control of UBC32-GFP and RFP was the internal control.
(F) ubc32atos9 double mutants partially rescue the insensitive phenotype of ubc32 to 150 mM NaCl. The picture was taken after growing for 10 days. Bar, 0.5 cm.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions