title: Wheat germ-based protein libraries for the functional characterisation of the Arabidopsis E2 ubiquitin conjugating enzymes and the RING-type E3 ubiquitin ligase enzymes.
abstract: BACKGROUND
  Protein ubiquitination is a ubiquitous mechanism in eukaryotes. In Arabidopsis, ubiquitin modification is mainly mediated by two ubiquitin activating enzymes (E1s), 37 ubiquitin conjugating enzymes (E2s), and more than 1300 predicted ubiquitin ligase enzymes (E3s), of which ~470 are RING-type E3s. A large proportion of the RING E3's gene products have yet to be characterised in vitro, likely because of the laborious work involved in large-scale cDNA cloning and protein expression, purification, and characterisation. In addition, several E2s, which might be necessary for the activity of certain E3 ligases, cannot be expressed by Escherichia coli or cultured insect cells and, therefore, remain uncharacterised.
  
  
  RESULTS
  Using the RIKEN Arabidopsis full-length cDNA library (RAFL) with the 'split-primer' PCR method and a wheat germ cell-free system, we established protein libraries of Arabidopsis E2 and RING E3 enzymes. We expressed 35 Arabidopsis E2s including six enzymes that have not been previously expressed, and 204 RING proteins, most of which had not been functionally characterised. Thioester assays using dithiothreitol (DTT) showed DTT-sensitive ubiquitin thioester formation for all E2s expressed. In expression assays of RING proteins, 31 proteins showed high molecular smears, which are probably the result of their functional activity. The activities of another 27 RING proteins were evaluated with AtUBC10 and/or a group of different E2s. All the 27 RING E3s tested showed ubiquitin ligase activity, including 17 RING E3s. Their activities are reported for the first time.
  
  
  CONCLUSIONS
  The wheat germ cell-free system used in our study, which is a eukaryotic expression system and more closely resembles the endogenous expression of plant proteins, is very suitable for expressing Arabidopsis E2s and RING E3s in their functional form. In addition, the protein libraries described here can be used for further understanding E2-E3 specificities and as platforms for protein-protein interaction screening.
authors.affiliation: Proteo-Science Center, Ehime University, Matsuyama, 790-8577, Japan. z862004m@mails.cc.ehime-u.ac.jp.|Botany Department, Faculty of Science, Ain Shams University, Cairo, 11566, Egypt. z862004m@mails.cc.ehime-u.ac.jp.
authors.forename: Abdelaziz
authors.initials: A
authors.lastname: Ramadan
authors.affiliation: Proteo-Science Center, Ehime University, Matsuyama, 790-8577, Japan. nemoto.keiichiro.mj@ehime-u.ac.jp.
authors.forename: Keiichirou
authors.initials: K
authors.lastname: Nemoto
authors.affiliation: Plant Genomic Network Research Team, RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan. motoaki.seki@riken.jp.|CREST, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan. motoaki.seki@riken.jp.
authors.forename: Motoaki
authors.initials: M
authors.lastname: Seki
authors.affiliation: Gene Discovery Research Group, RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan. kazuo.shinozaki@riken.jp.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Shinozaki
authors.affiliation: Proteo-Science Center, Ehime University, Matsuyama, 790-8577, Japan. takeda.hiroyuki.mk@ehime-u.ac.jp.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Takeda
authors.affiliation: Proteo-Science Center, Ehime University, Matsuyama, 790-8577, Japan. takahashi.hirotaka.rn@ehime-u.ac.jp.
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Takahashi
authors.affiliation: Proteo-Science Center, Ehime University, Matsuyama, 790-8577, Japan. sawasaki@ehime-u.ac.jp.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Sawasaki
mesh_terms: D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D018744:DNA, Plant; D015723:Gene Library; D010940:Plant Proteins / Q000235:genetics*; D014908:Triticum; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics* / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics* / Q000378:metabolism
chemical_list: D018744:DNA, Plant; D010940:Plant Proteins; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases