1. Volume 9, Issue 5, Pages 760-763 (May 2016)
AtBRO1 Functions in ESCRT-I Complex to Regulate Multivesicular Body Protein Sorting 
Jinbo Shen, Caiji Gao, Qiong Zhao, Youshun Lin, Xiangfeng Wang, Xiaohong Zhuang, Liwen Jiang 
Molecular Plant 
Volume 9, Issue 5, Pages (May 2016)
DOI: /j.molp
Copyright © 2016 The Author Terms and Conditions

2. Figure 1
The Arabidopsis ESCRT-I Protein AtBRO1 Functions in Multivesicular Body Protein Sorting.
(A) Colocalization of mCherry-AtBRO1 with GFP-FREE1 in punctate dots in root epidermal cells of 5-day-old Arabidopsis seedlings. The region indicated with the white rectangle is enlarged and shown on the right (from top to bottom: merged, mCherry-AtBRO1, and GFP-FREE1). Scale bar, 10 μm.
(B) Immunoprecipitation (IP) assay shows association between AtBRO1 and FREE1. Arabidopsis protoplasts expressing EYFP (lanes 1 and 3) or EYFP-AtBRO1 (lanes 2 and 4) with 3× Myc-FREE1 were subjected to protein extraction and IP with GFP-trap followed by immunoblotting with indicated antibodies.
(C) Immunoprecipitation (IP) assay shows association between AtBRO1 and FREE1 with Vps23A/ELC. Arabidopsis protoplasts expressing EYFP (lanes 1 and 3) or EYFP-Vps23A/ELC (lanes 2 and 4) with 3× Myc-FREE1 and 3× HA-AtBRO1 were subjected to protein extraction and IP with GFP-trap followed by immunoblotting with indicated antibodies.
(D and E) Acceptor photobleaching-fluorescence resonance energy transfer (FRET-AB) analysis and domain structures of AtBRO1 and FREE1. Ceru-AtBRO1 and EYFP-FREE1 or their deletion mutants were coexpressed in Arabidopsis protoplasts followed by confocal imaging (D, preventative confocal images) and FRET-AB quantifications (E, lower panel). The method to quantify FRET efficiency is described in Supplemental Materials and Methods. For each group, at least 10 individual protoplasts were used for FRET efficiency quantification and statistical analysis. Scale bar, 10 μm.
(F) A hypothetical model for AtBRO1 function in ESCRT-I complex: AtBRO1 recognizes and sorts ubiquitinated cargos into the ILVs of MVB for vacuolar degradation; AtBRO1-AMSH1 fusion protein deubiquitinates cargo and blocks subsequent sorting into the MVB pathway, leading to the tonoplast localization of cargo. In ESCRT-III complex, AtBRO1 binds to SNF7 and recruits DUb enzyme AMSH3 to deubiquitinate cargo. The Vps24-AMSH1 fusion protein did not affect cargo sorting into the vacuole.
(G–I) Expression of AtBRO1-AMSH1 fusion protein altered the localization of the membrane cargo protein EMP12-GFP on the tonoplast. Coexpression of HA-AtBRO1 (G), HA-AtBRO1-AMSH1 (H), or HA-Vps24-AMSH1 (I) with the membrane cargo proteins EMP12-GFP (green) and the tonoplast marker mRFP-VIT1 (red) in Arabidopsis protoplasts. Note that the degraded membrane proteins EMP12-GFP localized on the tonoplast (arrows) when coexpressing with HA-AtBRO1-AMSH1, instead of transporting into the vacuolar lumen. V, vacuole. Scale bar, 10 μm.
(J) AtBRO1 regulates the formation of the vacuole. Confocal microscopic images of the vacuolar membrane marker YFP-VAMP711 expressed in the matured embryo cells of wild-type or atbro1-1 mutant. Scale bars, 10 μm.
(K) AtBRO1 is essential for formation of ILVs in MVBs. Ultrathin sections were prepared from high-pressure frozen/freeze-substituted samples of roots of the DEX-inducible AtBRO1 RNAi plants without (−) or with (+) DEX induction, followed by immunogold labeling using VSR antibodies. The number of ILVs per MVB was statistically analyzed on 50 MVBs recognized by VSR antibodies from each sample. Scale bar, 100 nm.
(L) AtBRO1 regulates vacuolar sorting of PIN1-GFP. PIN1-GFP signal was detected in PM and tonoplast (arrows) in atbro1 mutant embryo compared with the PM localized signal in wild-type embryo. Scale bars, 10 μm.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions