title: Identification of downstream components of ubiquitin-conjugating enzyme PHOSPHATE2 by quantitative membrane proteomics in Arabidopsis roots.
abstract: MicroRNA399-mediated regulation of the ubiquitin-conjugating enzyme UBC24/phosphate2 (PHO2) is crucial for Pi acquisition and translocation in plants. Because of a potential role for PHO2 in protein degradation and its association with membranes, an iTRAQ (for isobaric tags for relative and absolute quantitation)- based quantitative membrane proteomic method was employed to search for components downstream of PHO2. A total of 7491 proteins were identified from Arabidopsis thaliana roots by mass spectrometry, 35.2% of which were predicted to contain at least one transmembrane helix. Among the quantifiable proteins, five were significantly differentially expressed between the wild type and pho2 mutant under two growth conditions. Using immunoblot analysis, we validated the upregulation of several members in phosphate transporter1 (PHT1) family and phosphate transporter traffic facilitator1 (PHF1) in pho2 and demonstrated that PHO2 mediates the degradation of PHT1 proteins. Genetic evidence that loss of PHF1 or PHT1;1 alleviated Pi toxicity in pho2 further suggests that they play roles as downstream components of PHO2. Moreover, we showed that PHO2 interacts with PHT1s in the postendoplasmic reticulum compartments and mediates the ubiquitination of endomembrane-localized PHT1;1. This study not only uncovers a mechanism by which PHO2 modulates Pi acquisition by regulating the abundance of PHT1s in the secretory pathway destined for plasma membranes, but also provides a database of the membrane proteome that will be widely applicable in root biology research.
authors.affiliation: Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Teng-Kuei
authors.initials: TK
authors.lastname: Huang
authors.forename: Chia-Li
authors.initials: CL
authors.lastname: Han
authors.forename: Shu-I
authors.initials: SI
authors.lastname: Lin
authors.forename: Yu-Ju
authors.initials: YJ
authors.lastname: Chen
authors.forename: Yi-Chuan
authors.initials: YC
authors.lastname: Tsai
authors.forename: Yet-Ran
authors.initials: YR
authors.lastname: Chen
authors.forename: June-Wei
authors.initials: JW
authors.lastname: Chen
authors.forename: Wei-Yi
authors.initials: WY
authors.lastname: Lin
authors.forename: Pei-Mien
authors.initials: PM
authors.lastname: Chen
authors.forename: Tzu-Yin
authors.initials: TY
authors.lastname: Liu
authors.forename: Ying-Shin
authors.initials: YS
authors.lastname: Chen
authors.forename: Ching-Mei
authors.initials: CM
authors.lastname: Sun
authors.forename: Tzyy-Jen
authors.initials: TJ
authors.lastname: Chiou
mesh_terms: D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000201:enzymology; D018506:Gene Expression Regulation, Plant; D006056:Golgi Apparatus / Q000201:enzymology; D028061:Phosphate Transport Proteins / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D018517:Plant Roots / Q000201:enzymology*; D025941:Protein Interaction Mapping; D059748:Proteolysis; D020543:Proteome / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D029681:Arabidopsis Proteins; C520845:PHT1;1 protein, Arabidopsis; D028061:Phosphate Transport Proteins; D010710:Phosphates; D020543:Proteome; C514696:UBC24 protein, Arabidopsis; D044763:Ubiquitin-Conjugating Enzymes